Methods of identifying candidate compounds of the human G protein-coupled receptor, GPR50, as modulators of body mass or adiposity

ABSTRACT

The present invention relates to methods of using a G protein-coupled receptor (GPCR) to screen one or more candidate compounds as a modulator of body mass or of adiposity or of percentage body fat in a subject or as a pharmaceutical agent for obesity and conditions related thereto. Inverse agonists and antagonists of the invention are useful as therapeutic agents for the prevention or treatment of obesity and conditions related thereto, including hypertension, insulin resistance, metabolic syndrome, Type 2 diabetes, dyslipidemia, atherosclerosis, coronary heart disease, and stroke. Agonists and partial agonists of the invention are useful as therapeutic agents for the prevention or treatment of disorders ameliorated by increasing body mass including, but not limited to, cachexia.

FIELD OF THE INVENTION

The present invention relates to methods of using a G protein-coupledreceptor (GPCR) to screen one or more candidate compounds as a modulatorof body mass or of adiposity or of percentage body fat in a subject oras a pharmaceutical agent for obesity and conditions related thereto.Inverse agonists and antagonists of the invention are useful astherapeutic agents for the prevention or treatment of obesity andconditions related thereto, including hypertension, insulin resistance,metabolic syndrome, Type 2 diabetes, dyslipidemia, atherosclerosis,coronary heart disease, and stroke. Agonists and partial agonists of theinvention are useful as therapeutic agents for the prevention ortreatment of disorders ameliorated by increasing body mass including,but not limited to, cachexia.

BACKGROUND OF THE INVENTION

The following discussion is intended to facilitate the understanding ofthe invention, but is not intended nor admitted to be prior art to theinvention.

A. Obesity

Obesity, which is defined as increased mass of adipose tissue, confers ahigher risk of cardiovascular and metabolic disorders such as Type 2diabetes, hyperlipidemia, and coronary heart disease and an associatedmorbidity and mortality. Metabolic syndrome, a multiplex risk factor forcardiovascular disease, is defined on the basis of five criteriaincluding one related to obesity [Grundy et al, Circulation (2004)109:433-438].

Obesity is now a major healthcare issue in the Western World andincreasingly in some third world countries. The increase in numbers ofobese people is due largely to the increasing preference for high fatcontent foods but also, and this can be a more important factor, thedecrease in activity in most people's lives. In the last 10 years therehas been a 30% increase in the incidence of obesity in the USA and thatabout 30% of the population of the USA is now considered obese.

Whether someone is classified as overweight or obese is generallydetermined on the basis of their body mass index (BMI) which iscalculated by dividing body weight (kg) by height squared (m²). Thus,the units of BMI are kg/m² and it is possible to calculate the BMI rangeassociated with minimum mortality in each decade of life. Overweight isdefined as a BMI in the range 25.0-29.9 kg/m², and obesity as a BMI of30 kg/m² or greater (see Table A below).

TABLE A CLASSIFICATION OF WEIGHT BY BODY MASS INDEX (BMI) BMICLASSIFICATION <18.5 Underweight 18.5-24.9 Normal 25.0-29.9 Overweight30.0-34.9 Obesity (Class I) 35.0-39.9 Obesity (Class II) >40 ExtremeObesity (Class III)

As the BMI increases there is an increased risk of death from a varietyof causes that is independent of other risk factors. The most commondiseases with obesity are cardiovascular disease particularlyhypertension), diabetes (obesity aggravates the development ofdiabetes), gallbladder disease, cancer and diseases of reproduction.Research has shown that even a modest reduction in body weight cancorrespond to a significant reduction in the risk of developing coronaryheart disease.

There are problems however with the BMI definition in that it does nottake into account the proportion of body mass that is muscle in relationto fat (adipose tissue). To account for this, obesity can also bedefined on the basis of body fat content: greater than 25% in males andgreater than 30% in females.

Obesity considerably increases the risk of developing cardiovasculardiseases as well. Coronary insufficiency, atheromatous disease, andcardiac insufficiency are at the forefront of the cardiovascularcomplication induced by obesity. It is estimated that if the entirepopulation had an ideal weight, the risk of coronary insufficiency woulddecrease by 25% and the risk of cardiac insufficiency and of cerebralvascular accidents by 35%. The incidence of coronary diseases is doubledin subjects less than 50 years of age who are 30% overweight. Thediabetes patient faces a 30% reduced lifespan. After age 45, people withdiabetes are about three times more likely than people without diabetesto have significant heart disease and up to five times more likely tohave a stroke. These findings emphasize the inter-relations betweenrisks factors for Type 2 diabetes and coronary heart disease and thepotential value of an integrated approach to the prevention of theseconditions based on the prevention of obesity [Perry, et al, BMJ (1995)310:560-564].

Diabetes has also been implicated in the development of kidney disease,eye diseases and nervous-system problems. Kidney disease, also callednephropathy, occurs when the kidney's “filter mechanism” is damaged andprotein leaks into urine in excessive amounts and eventually the kidneyfails. Diabetes is also a leading cause of damage to the retina at theback of the eye and increases risk of cataracts and glaucoma. Finally,diabetes is associated with nerve damage, especially in the legs andfeet, which interferes with the ability to sense pain and contributes toserious infections. Taken together, diabetes complications are one ofthe nation's leading causes of death.

The first line of treatment is to offer diet and life style advice topatients such as reducing the fat content of their diet and increasingtheir physical activity. However many patients find this difficult andneed additional help from drug therapy to maintain results from theseefforts.

Most currently marketed products have been unsuccessful as treatmentsfor obesity owing to a lack of efficacy or unacceptable side-effectprofiles. The most successful drug so far was the indirectly acting5-hydroxytryptamine (5-HT) agonist d-fenfluramine (Redux™) but reportsof cardiac valve defects in up to one third of patients led to itswithdrawal by the FDA in 1998.

In addition, two drugs have recently been launched in the USA andEurope: Orlistat (Xenical™), a drug that prevents absorption of fat bythe inhibition of pancreatic lipase, and Sibutramine (Reductil™), a5-HT/noradrenaline re-uptake inhibitor. However, side effects associatedwith these products may limit their long-term utility. Treatment withXenical™ is reported to induce gastrointestinal distress in somepatients, while Sibutramine has been associated with raised bloodpressure in some patients.

There is an unmet medical need for agents that safely decrease bodyweight. The present invention is directed to this, as well as other,important end.

B. GPR50

GPR50 is an orphan GPCR closely related to the G protein-coupledmelatonin receptor family. The gene for GPR50 is situated on the Xchromosome. Expression of GPR50 is reported to be restricted tohypothalamus and pituitary [Reppert et al, FEBS Letters (1996)386:219-224]. The coding region for GPR50 spans two exons. Several aminoacid polymorphisms of human GPR50 have been described; by reference toGenBank® Accession No. AAI03697, these include the substitutionsThr532Ala, Val606Ile, and the deletion Δ502-505 of four amino acids(Tbr.Thr.Gly.His).

C. G Protein-Coupled Receptors

Although a number of receptor classes exist in humans, by far the mostabundant and therapeutically relevant is represented by the Gprotein-coupled receptor (GPCR) class. It is estimated that there aresome 30,000-40,000 genes within the human genome, and of these,approximately 2% are estimated to code for GPCRs.

GPCRs represent an important area for the development of pharmaceuticalproducts: from approximately 20 of the 100 known GPCRs, approximately60% of all prescription pharmaceuticals have been developed. Forexample, in 1999, of the top 100 brand name prescription drugs, thefollowing drugs interact with GPCRs (the primary diseases and/ordisorders treated related to the drug is indicated in parentheses):

Claritin ® (allergies) Prozac ® (depression) Vasotec ® (hypertension)Paxil ® (depression) Zoloft ® (depression) Zyprexa ® (psychoticdisorder) Cozaar ® (hypertension) Imitrex ® (migraine) Zantac ® (reflux)Propulsid ® (reflux disease) Risperdal ® (schizophrenia) Serevent ®(asthma) Pepcid ® (reflux) Gaster ® (ulcers) Atrovent ® (bronchospasm)Effexor ® (depression) Depakote ® (epilepsy) Cardura ® (prostatichypertrophy) Allegra ® (allergies) Lupron ® (prostate cancer) Zoladex ®(prostate cancer) Diprivan ® (anesthesia) BuSpar ® (anxiety) Ventolin ®(bronchospasm) Hytrin ® (hypertension) Wellbutrin ® (depression)Zyrtec ® (rhinitis) Plavix ® (MI/stroke) Toprol-XL ® (hypertension)Tenormin ® (angina) Xalatan ® (glaucoma) Singulair ® (asthma) Diovan ®(hypertension) Harnal ® (prostatic hyperplasia) (Med Ad News 1999 Data).

GPCRs share a common structural motif, having seven sequences of between22 to 24 hydrophobic amino acids that form seven alpha helices, each ofwhich spans the membrane (each span is identified by number, i.e.,transmembrane-1 (TM-1), transmembrane-2 (TM-2), etc.). The transmembranehelices are joined by strands of amino acids between transmembrane-2 andtransmembrane-3, transmembrane-4 and transmembrane-5, andtransmembrane-6 and transmembrane-7 on the exterior, or “extracellular”side, of the cell membrane (these are referred to as “extracellular”regions 1, 2 and 3 (EC-1, EC-2 and EC-3), respectively). Thetransmembrane helices are also joined by strands of amino acids betweentransmembrane-1 and transmembrane-2, transmembrane-3 andtransmembrane-4, and transmembrane-5 and transmembrane-6 on theinterior, or “intracellular” side, of the cell membrane (these arereferred to as “intracellular” regions 1, 2 and 3 (IC-1, IC-2 and IC-3),respectively). The “carboxy” (“C”) terminus of the receptor lies in theintracellular space within the cell, and the “amino” (“N”) terminus ofthe receptor lies in the extracellular space outside of the cell.

Generally, when a ligand binds with the receptor (often referred to as“activation” of the receptor), there is a change in the conformation ofthe receptor that facilitates coupling between the intracellular regionand an intracellular “G-protein.” It has been reported that GPCRs are“promiscuous” with respect to G proteins, i.e., that a GPCR can interactwith more than one G protein. See, Kenakin, Life Sciences (1988)43:1095-1101. Although other G proteins exist, currently, Gq, Gs, Gi, Gzand Go are G proteins that have been identified. Ligand-activated GPCRcoupling with the G-protein initiates a signaling cascade process(referred to as “signal transduction”). Under normal conditions, signaltransduction ultimately results in cellular activation or cellularinhibition. Although not wishing to be bound to theory, it is thoughtthat the IC-3 loop as well as the carboxy terminus of the receptorinteract with the G protein.

Gs-coupled GPCRs elevate intracellular cAMP levels. GPCRs coupled to Gi,Go, or Gz lower intracellular cAMP levels. Gq-coupled GPCRs elevateintracellular IP₃ and Ca²⁺ levels.

There are also promiscuous G proteins, which appear to couple severalclasses of GPCRs to the phospholipase C pathway, such as G15 or G16[Offermanns & Simon, J Biol Chem (1995) 270:15175-80], or chimeric Gproteins designed to couple a large number of different GPCRs to thesame pathway, e.g. phospholipase C [Milligan & Rees, Trends inPharmaceutical Sciences (1999) 20:118-24]. A GPCR coupled to thephospholipase C pathway elevates intracellular IP₃ and Ca²⁺ levels.

Under physiological conditions, GPCRs exist in the cell membrane inequilibrium between two different conformations: an “inactive” state andan “active” state. A receptor in an inactive state is unable to link tothe intracellular signaling transduction pathway to initiate signaltransduction leading to a biological response. Changing the receptorconformation to the active state allows linkage to the transductionpathway (via the G-protein) and produces a biological response.

A receptor may be stabilized in an active state by a ligand or acompound such as a drug. Recent discoveries, including but notexclusively limited to modifications to the amino acid sequence of thereceptor, provide means other than ligands or drugs to promote andstabilize the receptor in the active state conformation. These meanseffectively stabilize the receptor in an active state by simulating theeffect of a ligand binding to the receptor. Stabilization by suchligand-independent means is termed “constitutive receptor activation.”

SUMMARY OF THE INVENTION

Nucleotide sequence encoding human GPR50 polypeptide is given in SEQ IDNO: 1; the amino acid sequence of said encoded human GPR50 polypeptideis given in SEQ ID NO: 2. Nucleotide sequence encoding human GPR50polypeptide comprising Δ502-505 is given in SEQ ID NO: 3; the amino acidsequence of said encoded human GPR50 polypeptide comprising Δ502-505 isgiven in SEQ ID NO: 4. Nucleotide sequence encoding mouse GPR50polypeptide is given in SEQ ID NO: 5; the amino acid sequence of saidencoded mouse GPR50 polypeptide is given in SEQ ID NO: 6. Nucleotidesequence encoding rat GPR50 polypeptide is given in SEQ ID NO: 7; theamino acid sequence of said encoded rat GPR50 polypeptide is given inSEQ ID NO: 8.

Applicants have shown that GPR50-deficient mice exhibit protection frombody weight gain induced by a high-fat diet. The present inventionfeatures methods relating to GPR50 for identifying a candidate compoundas a modulator of body mass or of adiposity or of percentage body fat ina subject or as a pharmaceutical agent for obesity and conditionsrelated thereto. Inverse agonists and antagonists of the invention areuseful as therapeutic agents for the prevention or treatment of obesityand conditions related thereto, including hypertension, insulinresistance, metabolic syndrome, Type 2 diabetes, dyslipidemia,atherosclerosis, coronary heart disease, and stroke.

In a first aspect, the invention features a method of identifying acandidate compound as a modulator of body mass in a subject, comprisingthe steps of:

-   -   (a) contacting the candidate compound with a GPCR comprising an        amino acid sequence selected from the group consisting of:        -   (i) the amino acid sequence of SEQ ID NO: 2;        -   (ii) amino acids 2-617 of SEQ ID NO: 2;        -   (iii) amino acids 2-617 of SEQ ID NO: 2, wherein the GPCR            does not comprise amino acids 1-617 of SEQ ID NO: 2;        -   (iv) the amino acid sequence of (i), (ii) or (iii), wherein            SEQ ID NO: 2 comprises any combination of a substitution of            serine at amino acid position 493 of SEQ ID NO: 2 with            asparagine, a substitution of threonine at amino acid            position 532 of SEQ ID NO: 2 with alanine, and a            substitution of valine at amino acid position 606 of SEQ ID            NO: 2 with isoleucine;        -   (v) the amino acid sequence of SEQ ID NO: 4;        -   (vi) amino acids 2-613 of SEQ ID NO: 4;        -   (vii) amino acids 2-613 of SEQ ID NO: 4, wherein the GPCR            does not comprise amino acids 1-613 of SEQ ID NO: 4;        -   (viii) the amino acid sequence of (v), (vi) or (vii),            wherein SEQ ID NO: 4 comprises any combination of a            substitution of asparagine at amino acid position 493 of SEQ            ID NO: 4 with serine, a substitution of alanine at amino            acid position 528 of SEQ ID NO: 4 with threonine, and a            substitution of valine at amino acid position 602 of SEQ ID            NO: 4 with isoleucine;        -   (ix) the amino acid sequence of a G protein-coupled receptor            encoded by a polynucleotide that is amplifiable by            polymerase chain reaction (PCR) on a human DNA sample using            specific primers SEQ ID NO: 9 and SEQ ID NO: 10;        -   (x) the amino acid sequence of a G protein-coupled receptor            encoded by a polynucleotide hybridizing at high stringency            to the complement of SEQ ID NO: 1 or SEQ ID NO:3;        -   (xi) the amino acid sequence of a G protein-coupled receptor            having at least about 75%, at least about 80%, at least            about 85%, at least about 90% or at least about 95% identity            to SEQ ID NO: 2 or SEQ ID NO: 4;        -   (xii) the amino acid sequence of SEQ ID NO: 6;        -   (xiii) amino acids 2-591 of SEQ ID NO: 6;        -   (xiv) amino acids 2-591 of SEQ ID NO: 6 wherein the GPCR            does not comprise amino acids 1-591 of SEQ ID NO: 6;        -   (xv) the amino acid sequence of a G protein-coupled receptor            encoded by a polynucleotide hybridizing at high stringency            to the complement of SEQ ID NO: 5;        -   (xvi) the amino acid sequence of a G protein-coupled            receptor having at least about 75%, at least about 80%, at            least about 85%, at least about 90% or at least about 95%            identity to SEQ ID NO: 6;        -   (xvii) the amino acid sequence of SEQ ID NO: 8;        -   (xviii) amino acids 2-594 of SEQ ID NO: 8;        -   (xix) amino acids 2-594 of SEQ ID NO: 8, wherein the GPCR            does not comprise amino acids 1-594 of SEQ ID NO: 8;        -   (xx) the amino acid sequence of a G protein-coupled receptor            encoded by a polynucleotide hybridizing at high stringency            to the complement of SEQ ID NO: 7;        -   (xxi) the amino acid sequence of a G protein-coupled            receptor having at least about 75%, at least about 80%, at            least about 85%, at least about 90% or at least about 95%            identity to SEQ ID NO: 8; and        -   (xxii) the amino acid sequence of a G protein-coupled            receptor that is a constitutively active version of a            receptor having SEQ ID NO: 2 or SEQ ID NO: 4;        -   or a variant or biologically active fragment thereof;            wherein the receptor couples to a G protein; and    -   (b) determining the ability of the compound to inhibit or        stimulate functionality of the GPCR,        wherein the ability of the compound to inhibit or stimulate        functionality of the GPCR is indicative of the compound being a        modulator of body mass in the subject.

In some embodiments, the GPCR comprises the amino acid sequence of SEQID NO: 2 or SEQ ID NO: 4.

In some embodiments, the GPCR comprises the amino acid sequence of a Gprotein-coupled receptor having at least about 75%, at least about 80%,at least about 85%, at least about 90% or at least about 95% identity toSEQ ID NO: 2 or SEQ ID NO: 4.

In some embodiments, the body mass comprises weight gain induced by ahigh fat diet.

In some embodiments, the subject is a mammal. In some embodiments, themarmmal is a human.

The invention also features a method of identifying a candidate compoundas a modulator of adiposity in a subject, comprising the steps of:

-   -   (a) contacting the candidate compound with a GPCR comprising an        amino acid sequence selected from the group consisting of:        -   (i) the amino acid sequence of SEQ ID NO: 2;        -   (ii) amino acids 2-617 of SEQ ID NO: 2;        -   (iii) amino acids 2-617 of SEQ ID NO: 2, wherein the GPCR            does not comprise amino acids 1-617 of SEQ ID NO: 2;        -   (iv) the amino acid sequence of (i), (ii) or (iii), wherein            SEQ ID NO: 2 comprises any combination of a substitution of            serine at amino acid position 493 of SEQ ID NO: 2 with            asparagine, a substitution of threonine at amino acid            position 532 of SEQ ID NO: 2 with alanine, and a            substitution of valine at amino acid position 606 of SEQ ID            NO: 2 with isoleucine;        -   (v) the amino acid sequence of SEQ ID NO: 4;        -   (vi) amino acids 2-613 of SEQ ID NO: 4;        -   (vii) amino acids 2-613 of SEQ ID NO: 4, wherein the GPCR            does not comprise amino acids 1-613 of SEQ ID NO: 4;        -   (viii) the amino acid sequence of (v), (vi) or (vii),            wherein SEQ ID NO: 4 comprises any combination of a            substitution of asparagine at amino acid position 493 of SEQ            ID NO: 4 with serine, a substitution of alanine at amino            acid position 528 of SEQ ID NO: 4 with threonine, and a            substitution of valine at amino acid position 602 of SEQ ID            NO: 4 with isoleucine;        -   (ix) the amino acid sequence of a G protein-coupled receptor            encoded by a polynucleotide that is amplifiable by            polymerase chain reaction (PCR) on a human DNA sample using            specific primers SEQ ID NO: 9 and SEQ ID NO: 10;        -   (x) the amino acid sequence of a G protein-coupled receptor            encoded by a polynucleotide hybridizing at high stringency            to the complement of SEQ ID NO: 1 or SEQ ID NO: 3;        -   (xi) the amino acid sequence of a G protein-coupled receptor            having at least about 75%, at least about 80%, at least            about 85%, at least about 90% or at least about 95% identity            to SEQ ID NO: 2 or SEQ ID NO: 4;        -   (xii) the amino acid sequence of SEQ ID NO: 6;        -   (xiii) amino acids 2-591 of SEQ ID NO: 6;        -   (xiv) amino acids 2-591 of SEQ ID NO: 6 wherein the GPCR            does not comprise amino acids 1-591 of SEQ ID NO: 6;        -   (xv) the amino acid sequence of a G protein-coupled receptor            encoded by a polynucleotide hybridizing at high stringency            to the complement of SEQ ID NO: 5;        -   (xvi) the amino acid sequence of a G protein-coupled            receptor having at least about 75%, at least about 80%, at            least about 85%, at least about 90% or at least about 95%            identity to SEQ ID NO: 6;        -   (xvii) the amino acid sequence of SEQ ID NO: 8;        -   (xviii) amino acids 2-594 of SEQ ID NO: 8;        -   (xix) amino acids 2-594 of SEQ ID NO: 8, wherein the GPCR            does not comprise amino acids 1-594 of SEQ ID NO: 8;        -   (xx) the amino acid sequence of a G protein-coupled receptor            encoded by a polynucleotide hybridizing at high stringency            to the complement of SEQ ID NO: 7;        -   (xxi) the amino acid sequence of a G protein-coupled            receptor having at least about 75%, at least about 80%, at            least about 85%, at least about 90% or at least about 95%            identity to SEQ ID NO: 8; and        -   (xxii) the amino acid sequence of a G protein-coupled            receptor that is a constitutively active version of a            receptor having SEQ ID NO: 2 or SEQ ID NO: 4;        -   or a variant or biologically active fragment thereof;            wherein the receptor couples to a G protein; and    -   (b) determining the ability of the compound to inhibit or        stimulate functionality of the GPCR;        wherein the ability of the compound to inhibit or stimulate        functionality of the GPCR is indicative of the compound being a        modulator of adiposity in the subject.

In some embodiments, the GPCR comprises the amino acid sequence of SEQID NO: 2 or SEQ ID NO: 4.

In some embodiments, the GPCR comprises the amino acid sequence of a Gprotein-coupled receptor having at least about 75%, at least about 80%,at least about 85%, at least about 90% or at least about 95% identity toSEQ ID NO: 2 or SEQ ID NO: 4.

In some embodiments, the adiposity comprises increased adiposity inducedby a high fat diet.

In some embodiments, the subject is a mammal. In some embodiments, themammal is a human.

The invention also features a method of identifying a candidate compoundas a modulator of percentage body fat in a subject, comprising the stepsof:

-   -   (a) contacting the candidate compound with a GPCR comprising an        amino acid sequence selected from the group consisting of:        -   (i) the amino acid sequence of SEQ ID NO: 2;        -   (ii) amino acids 2-617 of SEQ ID NO: 2;        -   (iii) amino acids 2-617 of SEQ ID NO: 2, wherein the GPCR            does not comprise amino acids 1-617 of SEQ ID NO: 2;        -   (iv) the amino acid sequence of (i), (ii) or (iii), wherein            SEQ ID NO: 2 comprises any combination of a substitution of            serine at amino acid position 493 of SEQ ID NO: 2 with            asparagine, a substitution of threonine at amino acid            position 532 of SEQ ID NO: 2 with alanine, and a            substitution of valine at amino acid position 606 of SEQ ID            NO: 2 with isoleucine;        -   (v) the amino acid sequence of SEQ ID NO: 4;        -   (vi) amino acids 2-613 of SEQ ID NO: 4;        -   (vii) amino acids 2-613 of SEQ ID NO: 4, wherein the GPCR            does not comprise amino acids 1-613 of SEQ ID NO: 4;        -   (viii) the amino acid sequence of (v), (vi) or (vii),            wherein SEQ ID NO: 4 comprises any combination of a            substitution of asparagine at amino acid position 493 of SEQ            ID NO: 4 with serine, a substitution of alanine at amino            acid position 528 of SEQ ID NO: 4 with threonine, and a            substitution of valine at amino acid position 602 of SEQ ID            NO: 4 with isoleucine;        -   (ix) the amino acid sequence of a G protein-coupled receptor            encoded by a polynucleotide that is amplifiable by            polymerase chain reaction (PCR) on a human DNA sample using            specific primers SEQ ID NO: 9 and SEQ ID NO: 10;        -   (x) the amino acid sequence of a G protein-coupled receptor            encoded by a polynucleotide hybridizing at high stringency            to the complement of SEQ ID NO: 1 or SEQ ID NO: 3;        -   (xi) the amino acid sequence of a G protein-coupled receptor            having at least about 75%, at least about 80%, at least            about 85%, at least about 90% or at least about 95% identity            to SEQ ID NO: 2 or SEQ ID NO: 4;        -   (xii) the amino acid sequence of SEQ ID NO: 6;        -   (xiii) amino acids 2-591 of SEQ ID NO: 6;        -   (xiv) amino acids 2-591 of SEQ ID NO: 6 wherein the GPCR            does not comprise amino acids 1-591 of SEQ ID NO: 6;        -   (xv) the amino acid sequence of a G protein-coupled receptor            encoded by a polynucleotide hybridizing at high stringency            to the complement of SEQ ID NO: 5;        -   (xvi) the amino acid sequence of a G protein-coupled            receptor having at least about 75%, at least about 80%, at            least about 85%, at least about 90% or at least about 95%            identity to SEQ ID NO: 6;        -   (xvii) the amino acid sequence of SEQ ID NO: 8;        -   (xviii) amino acids 2-594 of SEQ ID NO: 8;        -   (xix) amino acids 2-594 of SEQ ID NO: 8, wherein the GPCR            does not comprise amino acids 1-594 of SEQ ID NO: 8;        -   (xx) the amino acid sequence of a G protein-coupled receptor            encoded by a polynucleotide hybridizing at high stringency            to the complement of SEQ ID NO: 7;        -   (xxi) the amino acid sequence of a G protein-coupled            receptor having at least about 75%, at least about 80%, at            least about 85%, at least about 90% or at least about 95%            identity to SEQ ID NO: 8; and        -   (xxii) the amino acid sequence of a G protein-coupled            receptor that is a constitutively active version of a            receptor having SEQ ID NO: 2 or SEQ ID NO: 4;        -   or a variant or biologically active fragment thereof;            wherein the receptor couples to a G protein; and    -   (b) determining the ability of the compound to inhibit or        stimulate functionality of the GPCR;        wherein the ability of the compound to inhibit or stimulate        functionality of the GPCR is indicative of the compound being a        modulator of percentage body fat in the subject.

In some embodiments, the GPCR comprises the amino acid sequence of SEQID NO: 2 or SEQ ID NO: 4.

In some embodiments, the GPCR comprises the amino acid sequence of a Gprotein-coupled receptor having at least about 75%, at least about 80%,at least about 85%, at least about 90% or at least about 95% identity toSEQ ID NO: 2 or SEQ ID NO: 4.

In some embodiments, percentage body fat comprises increased percentagebody fat induced by a high fat diet.

In some embodiments, the subject is a mammal. In some embodiments, themammal is a human.

The invention also features a method of identifying a candidate compoundas a pharmaceutical agent for obesity or a condition related thereto,comprising the steps of:

-   -   (a′) contacting the candidate compound with a GPCR comprising an        amino acid sequence selected from the group consisting of:        -   (i) the amino acid sequence of SEQ ID NO: 2;        -   (ii) amino acids 2-617 of SEQ ID NO: 2;        -   (iii) amino acids 2-617 of SEQ ID NO: 2, wherein the GPCR            does not comprise amino acids 1-617 of SEQ ID NO: 2;        -   (iv) the amino acid sequence of (i), (ii) or (iii), wherein            SEQ ID NO: 2 comprises any combination of a substitution of            serine at amino acid position 493 of SEQ ID NO: 2 with            asparagine, a substitution of threonine at amino acid            position 532 of SEQ ID NO: 2 with alanine, and a            substitution of valine at amino acid position 606 of SEQ ID            NO: 2 with isoleucine;        -   (v) the amino acid sequence of SEQ ID NO: 4;        -   (vi) amino acids 2-613 of SEQ ID NO: 4;        -   (vii) amino acids 2-613 of SEQ ID NO: 4, wherein the GPCR            does not comprise amino acids 1-613 of SEQ ID NO: 4;        -   (viii) the amino acid sequence of (v), (vi) or (vii),            wherein SEQ ID NO: 4 comprises any combination of a            substitution of asparagine at amino acid position 493 of SEQ            ID NO: 4 with serine, a substitution of alanine at amino            acid position 528 of SEQ ID NO: 4 with threonine, and a            substitution of valine at amino acid position 602 of SEQ ID            NO: 4 with isoleucine;        -   (ix) the amino acid sequence of a G protein-coupled receptor            encoded by a polynucleotide that is amplifiable by            polymerase chain reaction (PCR) on a human DNA sample using            specific primers SEQ ID NO: 9 and SEQ ID NO: 10;        -   (x) the amino acid sequence of a G protein-coupled receptor            encoded by a polynucleotide hybridizing at high stringency            to the complement of SEQ ID NO: 1 or SEQ ID NO: 3;        -   (xi) the amino acid sequence of a G protein-coupled receptor            having at least about 75%, at least about 80%, at least            about 85%, at least about 90% or at least about 95% identity            to SEQ ID NO: 2 or SEQ ID NO: 4;        -   (xii) the amino acid sequence of SEQ ID NO: 6;        -   (xiii) amino acids 2-591 of SEQ ID NO: 6;        -   (xiv) amino acids 2-591 of SEQ ID NO: 6 wherein the GPCR            does not comprise amino acids 1-591 of SEQ ID NO: 6;        -   (xv) the amino acid sequence of a G protein-coupled receptor            encoded by a polynucleotide hybridizing at high stringency            to the complement of SEQ ID NO: 5;        -   (xvi) the amino acid sequence of a G protein-coupled            receptor having at least about 75%, at least about 80%, at            least about 85%, at least about 90% or at least about 95%            identity to SEQ ID NO: 6;        -   (xvii) the amino acid sequence of SEQ ID NO: 8;        -   (xviii) amino acids 2-594 of SEQ ID NO: 8;        -   (xix) amino acids 2-594 of SEQ ID NO: 8, wherein the GPCR            does not comprise amino acids 1-594 of SEQ ID NO: 8;        -   (xx) the amino acid sequence of a G protein-coupled receptor            encoded by a polynucleotide hybridizing at high stringency            to the complement of SEQ ID NO: 7;        -   (xxi) the amino acid sequence of a G protein-coupled            receptor having at least about 75%, at least about 80%, at            least about 85%, at least about 90% or at least about 95%            identity to SEQ ID NO: 8; and        -   (xxii) the amino acid sequence of a G protein-coupled            receptor that is a constitutively active version of a            receptor having SEQ ID NO: 2 or SEQ ID NO: 4;        -   or a variant or biologically active fragment thereof;            wherein the receptor couples to a G protein; and    -   (b′) determining the ability of the compound to inhibit        functionality of the GPCR; wherein the ability of the compound        to inhibit functionality of the GPCR is indicative of the        compound being a pharmaceutical agent for obesity or the        condition related thereto.

The invention additionally features a method of identifying a candidatecompound as a pharmaceutical agent for obesity or a condition relatedthereto, comprising steps (a′) and (b′) of this first aspect, andfurther comprising:

-   -   (c′) optionally synthesizing a compound which inhibits        functionality of the GPCR in step (b′);    -   (d′) administering a compound which inhibits functionality of        the GPCR in step (b′) to a mammal; and    -   (e′) determining whether the compound confers protection from        weight gain in the mammal;        wherein the ability of the candidate compound to confer        protection from weight gain in the mammal is indicative of the        candidate compound being a pharmaceutical agent for obesity or a        condition related thereto.

In some embodiments, said protection from weight gain in the mammalcomprises protection from weight gain in the mammal induced by a highfat diet.

In some embodiments, the candidate compound is shown to conferprotection from weight gain in the mammal induced by a high fat diet.

In some embodiments, the pharmaceutical agent for obesity or a conditionrelated thereto is a compound for preventing or treating obesity or acondition related thereto.

In some embodiments, the compound which inhibits functionality of theGPCR in step (b′) is an inverse agonist or antagonist of the GPCR.

In some embodiments, the mammal is a non-human mammal. In someembodiments, the mammal is a laboratory animal. In some embodiments, themammal is a non-human primate. In some embodiments, the mammal is arodent. In some embodiments, the mammal is a rat. In some embodiments,the mammal is a mouse.

In some embodiments, the GPCR comprises the amino acid sequence of SEQID NO: 2 or SEQ ID NO: 4.

In some embodiments, the GPCR comprises the amino acid sequence of a Gprotein-coupled receptor having at least about 75%, at least about 80%,at least about 85%, at least about 90% or at least about 95% identity toSEQ ID NO: 2 or SEQ ID NO: 4.

In some embodiments, the G protein-coupled receptor having at leastabout 75%, at least about 80%, at least about 85%, at least about 90% orat least about 95% identity to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6or SEQ ID NO: 8 is an endogenous GPCR. In some embodiments, the Gprotein-coupled receptor having at least about 75%, at least about 80%,at least about 85%, at least about 90% or at least about 95% identity toSEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8 is a mammalianendogenous GPCR. In some embodiments, the G protein-coupled receptorhaving at least about 75%, at least about 80%, at least about 85%, atleast about 90% or at least about 95% identity to SEQ ID NO: 2, SEQ IDNO: 4, SEQ ID NO: 6 or SEQ ID NO: 8 is a non-endogenous GPCR.

In some embodiments, the G protein-coupled receptor that is aconstitutively active version of a receptor having SEQ ID NO: 2 or SEQID NO: 4 is an endogenous G protein-coupled receptor. In someembodiments, the G protein-coupled receptor that is a constitutivelyactive version of a receptor having SEQ ID NO: 2 or SEQ ID NO: 4 is anon-endogenous G protein-coupled receptor.

In some embodiments, the obesity comprises weight gain induced by a highfat diet.

In some embodiments, the condition related to obesity is selected fromthe group consisting of hypertension, insulin resistance, metabolicsyndrome, Type 2 diabetes, dyslipidemia, atherosclerosis, coronary heartdisease, and stroke.

In some embodiments, the pharmaceutical agent for obesity or a conditionrelated thereto is a compound for preventing or treating obesity or acondition related thereto.

In some embodiments, the compound that inhibits functionality of theGPCR is an inverse agonist or antagonist of the GPCR. In someembodiments, the compound that inhibits functionality of the GPCR is aninverse agonist of the GPCR. In some embodiments, the compound thatinhibits functionality of the GPCR is an antagonist of the GPCR.

In some embodiments, the GPCR exhibits a detectable level ofconstitutive activity. In some embodiments, the constitutive activity isfor lowering a level of intracellular cAMP. In some embodiments, theconstitutive activity is for causing melanophore cells to undergopigment aggregation.

In some embodiments, said contacting comprises contacting in thepresence of a known agonist of the GPCR. In some embodiments, the knownagonist of the GPCR is a known agonist of endogenous human GPR50. Insome embodiments relating to said contacting comprising contacting inthe presence of a known agonist of the GPCR, the candidate compound iscontacted with the GPCR prior to the known agonist being contacted withthe GPCR. In some embodiments relating to said contacting comprisingcontacting in the presence of a known agonist of the GPCR, the candidatecompound is contacted with the GPCR for a period of up to severalminutes prior to the known agonist being contacted with the GPCR. Insome embodiments relating to said contacting comprising contacting inthe presence of a known agonist of the GPCR, the candidate compound iscontacted with the GPCR for a period of up to about 5 min, of up toabout 10 min or of up to about 30 min prior to the known agonist beingcontacted with the GPCR.

In some embodiments, said contacting comprises contacting in the absenceof a known ligand of the GPCR. In some embodiments, said contactingcomprises contacting in the absence of a known ligand of endogenoushuman GPR50. In some embodiments, said contacting comprises contactingin the absence of a known agonist of the GPCR. In some embodiments, saidcontacting comprises contacting in the absence of a known agonist ofendogenous human GPR50.

In some embodiments, PCR is RT-PCR.

In some embodiments, the human DNA is human cDNA derived from a tissueor cell type that expresses GPR50. In some embodiments, the human cDNAis derived from hypothalamus or pituitary.

In some embodiments, the G protein-coupled receptor encoded by apolynucleotide that is amplifiable by polymerase chain reaction (PCR) ona human DNA sample using specific primers SEQ ID NO: 9 and SEQ ID NO: 10is an endogenous GPR50 G protein-coupled receptor.

In some embodiments, the subject is a mammal. In some embodiments, thesubject is a mammal selected from the group consisting of mouse, rat andhuman. In some embodiments, the subject is a human.

In some embodiments, the subject is overweight or obese. In someembodiments, the subject is overweight. In some embodiments, the subjectis obese.

In some embodiments, the ability of the compound to inhibitfunctionality of the GPCR is indicative of the compound being a compoundthat decreases body mass in the subject.

In some embodiments, the ability of the compound to inhibitfunctionality of the GPCR is indicative of the compound being a compoundthat decreases adiposity in the subject.

In some embodiments, the ability of the compound to inhibitfunctionality of the GPCR is indicative of the compound being a compoundthat decreases percentage body fat in the subject.

In some embodiments, the condition related to obesity is selected fromthe group consisting of hypertension, congestive cardiomyopathy,varicosities, pulmonary embolism, coronary heart disease, stroke,idiopathic intracranial hypertension, meralgia parethetica, dyspnea,obstructive sleep apnea, hypoventilation syndrome, Pickwickian syndrome,asthma, immobility, degenerative osteoarthritis, low back pain, striaedistensae or “stretch marks,” venous stasis of the lower extremities,lymphedema, cellulitis, intertrigo, carbuncles, acanthosis nigricans,skin tags, gastro-esophageal reflux disorder, nonalcoholic fattyliver/steatohepatitis, cholelithiasis, hernias, colon cancer, stressincontinence, obesity-related glomerulopathy, breast and uterine cancer,depression and low self-esteem, impaired quality of life, metabolicsyndrome, insulin resistance, Type 2 diabetes, dyslipidemia,atherosclerosis, hyperandrogenemia in women, polycystic ovariansyndrome, dysmenorrhea, infertility, pregnancy complications, and malehypogonadism. In some embodiments, the condition related to obesity isselected from the group consisting of hypertension, insulin resistance,metabolic syndrome, Type 2 diabetes, dyslipidemia, atherosclerosis,coronary heart disease, and stroke.

In some embodiments, the GPCR is recombinant.

In some embodiments, the GPCR is endogenous. In some embodiments, theGPCR that is endogenous is a mammalian endogenous GPCR. In someembodiments, the GPCR that is a mammalian endogenous GPCR is a mammalianendogenous GPR50. In some embodiments, the GPCR is non-endogenous.

In some embodiments, the GPCR is a mammalian GPR50.

In some embodiments, the G protein-coupled receptor encoded by apolynucleotide hybridizing at high stringency to the complement of SEQID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7 is an endogenousGPCR. In some embodiments, the G protein-coupled receptor encoded by apolynucleotide hybridizing at high stringency to the complement of SEQID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7 that is anendogenous GPCR is a mammalian GPCR. In some embodiments, the Gprotein-coupled receptor encoded by a polynucleotide hybridizing at highstringency to the complement of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5or SEQ ID NO: 7 exhibits a detectable level of constitutive activity. Insome embodiments, the constitutive activity is for lowering a level ofintracellular cAMP. In some embodiments, the constitutive activity isfor causing melanophore cells to undergo pigment aggregation. In certainembodiments, the G protein-coupled receptor encoded by a polynucleotidehybridizing at high stringency to the complement of SEQ ID NO: 1, SEQ IDNO: 3, SEQ ID NO: 5 or SEQ ID NO: 7 specifically binds an antibody thatrecognizes an mammalian endogenous GPR50 (an antibody that recognizes anendogenous mammalian GPR50 can be obtained commercially from, e.g.,Advanced Targeting Systems, San Diego, Calif.; and CHEMICONInternational, Inc., Temecula, Calif.) or specifically binds a knownligand of an mammalian endogenous GPR50. In certain embodiments, theknown ligand of the mammalian endogenous GPR50 is an endogenous ligandof the mammalian endogenous GPR50.

In some embodiments, the G protein-coupled receptor having at leastabout 75% identity, at least about 80%, at least about 85%, at leastabout 90% or at least about 95% identity to SEQ ID NO: 2 or SEQ ID NO: 4is an endogenous G protein-coupled receptor. In some embodiments, the Gprotein-coupled receptor having at least about 75% identity, at leastabout 80%, at least about 85%, at least about 90% Or at least about 95%identity to SEQ ID NO: 2 or SEQ ID NO: 4 is a non-endogenous Gprotein-coupled receptor. In some embodiments, the G protein-coupledreceptor having at least about 75% identity, at least about 80%, atleast about 85%, at least about 90% or at least about 95% identity toSEQ ID NO: 6 is an endogenous G protein-coupled receptor. In someembodiments, the G protein-coupled receptor having at least about 75%identity, at least about 80%, at least about 85%, at least about 90% orat least about 95% identity to SEQ ID NO: 6 is a non-endogenous Gprotein-coupled receptor. In some embodiments, the G protein-coupledreceptor having at least about 75% identity, at least about 80%, atleast about 85%, at least about 90% or at least about 95% identity toSEQ ID NO: 8 is an endogenous G protein-coupled receptor. In someembodiments, the G protein-coupled receptor having at least about 75%identity, at least about 80%, at least about 85%, at least about 90% orat least about 95% identity to SEQ ID NO: 8 is a non-endogenous Gprotein-coupled receptor.

In some embodiments, said contacting comprises contacting the candidatecompound with a host cell or with membrane of a host cell that expressesthe GPCR, wherein said host cell comprises an expression vectorcomprising a polynucleotide encoding the GPCR.

In some embodiments, the method comprises co-transfecting a host cellwith the GPCR and with thyroid-stimulating hormone receptor (TSHR).

In some embodiments, said determining is carried out with membranecomprising the GPCR.

In some embodiments, the method comprises detecting a second messenger.

In some embodiments, said determining is by a process comprising themeasurement of a level of a second messenger selected from the groupconsisting of cyclic AMP (cAMP), cyclic GMP (cGMP), inositol1,4,5-triphosphate (IP₃), diacylglycerol (DAG), MAP kinase activity,MAPK/ERK kinase kinase-1 (MEKK1) activity, and Ca²⁺. In someembodiments, said second messenger is cAMP. In some embodiments, thelevel of intracellular cAMP is increased.

In some embodiments, said determining is by a process comprising the useof a Melanophore assay. In some embodiments, the melanophore cellsundergo pigment aggregation. In some embodiments, the candidate compoundinhibits agonist induced pigment aggregation. In some embodiments, thecandidate compound inhibits a level of constitutively induced pigmentaggregation.

In some embodiments, said determining is by a process comprising themeasurement of GTPγS binding to membrane comprising the GPCR. In someembodiments, GTPγS binding to membrane comprising the GPCR is decreased.

In some embodiments, the modulator of body mass or adiposity orpercentage body fat in the subject is a modulator of the GPCR selectedfrom the group consisting of agonist, partial agonist, inverse agonistand antagonist. In some embodiments, the modulator of body mass oradiposity or percentage body fat in the subject is an inverse agonist orantagonist of the GPCR. In some embodiments, the modulator of body massor adiposity or percentage body fat in the subject is an inverse agonistof the GPCR. In some embodiments, the modulator of body mass oradiposity or percentage body fat in the subject is an antagonist of theGPCR. In some embodiments, the modulator of body mass or adiposity orpercentage body fat in the subject is a modulator of a mammalian GPR50.In some embodiments, the modulator of body mass or adiposity orpercentage body fat in the subject is a modulator of a human GPR50.

In some embodiments, the candidate compound is a small molecule.

In some embodiments, the candidate compound is a polypeptide. In someembodiments, the candidate compound is not an antibody or anantigen-binding fragment thereof. In some embodiments, the candidatecompound is a polypeptide, provided that the polypeptide is not anantibody or an antigen-binding fragment thereof. In some embodiments,the candidate compound is an antibody or an antigen-binding fragmentthereof. In some embodiments, the candidate compound is a lipid. In someembodiments, the candidate compound is not a polypeptide. In someembodiments, the candidate compound is not a lipid. In some embodiments,the candidate compound is non-endogenous. In some embodiments, thecandidate compound is not endogenous. In some embodiments, the candidatecompound is not material that a prokaryote or eukaryote naturallyproduces. In some embodiments, the candidate compound is not materialthat a prokaryote naturally produces. In some embodiments, the candidatecompound is not material that a eukaryote naturally produces. In someembodiments, the candidate compound is not material that a mammalnaturally produces. In some embodiments, the candidate compound is acompound not known to inhibit or stimulate functionality of the GPCR. Insome embodiments, the candidate compound is a compound not known to bean agonist of the GPCR. In some embodiments, the candidate compound is acompound not known to be a partial agonist of the GPCR. In someembodiments, the candidate compound is a compound not known to be aninverse agonist of the GPCR. In some embodiments, the candidate compoundis a compound not known to be an antagonist of the GPCR.

In some embodiments, the modulator of body mass or adiposity orpercentage body fat in the subject is an agonist, partial agonist,inverse agonist or antagonist of the GPCR. In some embodiments, themodulator of body mass or adiposity or percentage body fat in thesubject is an inverse agonist or antagonist of the GPCR.

In some embodiments, the pharmaceutical agent is an inverse agonist orantagonist of the GPCR.

In some embodiments, the method further comprises the step of comparingthe modulation of the receptor caused by the candidate compound to asecond modulation of the receptor caused by contacting the receptor witha known modulator of the receptor.

In some embodiments, said method further comprises the step offormulating the modulator or the pharmaceutical agent into apharmaceutical composition. In some embodiments, the modulator or thepharmaceutical agent is an inverse agonist or an antagonist of the GPCR.

In some embodiments, said method further comprises synthesis of themodulator or the pharmaceutical agent. In some embodiments, themodulator or the pharmaceutical agent is an inverse agonist or anantagonist of the GPCR.

In some embodiments, said method further comprises: optionally,determining the structure of the modulator or the pharmaceutical agent;and providing the modulator or the pharmaceutical agent or the name orstructure of the modulator or the pharmaceutical agent. In someembodiments, the modulator or the pharmaceutical agent is an inverseagonist or an antagonist of the GPCR.

In some embodiments, said method further comprises: optionally,determining the structure of the modulator or the pharmaceutical agent;optionally, providing the modulator or the pharmaceutical agent or thename or structure of the modulator or the pharmaceutical agent; andproducing or synthesizing the modulator or the pharmaceutical agent. Insome embodiments, the modulator or the pharmaceutical agent is aninverse agonist or an antagonist of the GPCR.

In some embodiments, the method comprises identifying an agonist of theGPCR. In some embodiments, the method further comprises formulating saidagonist as a pharmaceutical. In some embodiments, the method comprisesidentifying a partial agonist of the GPCR. In some embodiments, themethod further comprises formulating said partial agonist as apharmaceutical. In some embodiments, the method comprises identifying aninverse agonist of the GPCR. In some embodiments, the method furthercomprises formulating said inverse agonist as a pharmaceutical. In someembodiments, the method comprises identifying an antagonist of the GPCR.In some embodiments, the method further comprises formulating saidantagonist as a pharmaceutical.

In some embodiments, the baseline intracellular response (e.g., theresponse in the absence of a known agonist) is inhibited in the presenceof the inverse agonist by at least about 10%, at least about 20%, atleast about 30%, at least about 40%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, or at least about 95% as comparedwith the baseline response in the absence of the inverse agonist.

In some embodiments, the baseline intracellular response (e.g., theresponse in the presence of a known agonist) is inhibited in thepresence of the antagonist by at least about 10%, at least about 20%, atleast about 30%, at least about 40%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, or at least about 95% as comparedwith the baseline response in the absence of the antagonist.

In some embodiments, said contacting of the candidate compound with aGPCR comprises contacting the candidate compound with a eukaryotic hostcell comprising the GPCR or with membrane thereof comprising the GPCR.In some embodiments, the eukaryotic host cell is a mammalian host cell.In some embodiments, the mammalian host cell is a CHO cell, a COS-7cell, an MCB3901 cell, a 293 cell or a 293T cell. In some embodiments,the eukaryotic host cell is a melanophore host cell. In someembodiments, the eukaryotic host cell is a yeast host cell. In someembodiments, the eukaryotic host cell is a recombinant eukaryotic hostcell.

In a second aspect, the invention features a modulator or apharmaceutical agent identifiable according to a method of the firstaspect.

In some embodiments, the modulator or the pharmaceutical agent isidentified according to a method of the first aspect.

In some embodiments, the modulator or the pharmaceutical agent is anagonist, a partial agonist, an inverse agonist, or an antagonist of theGPCR. In some embodiments, the agonist, partial agonist, inverseagonist, or antagonist of the GPCR is an agonist, partial agonist,inverse agonist, or antagonist of a mammalian GPR50. In someembodiments, the mammalian GPR50 is a human GPR50.

In some embodiments, the modulator or the pharmaceutical agent is aninverse agonist or antagonist of the GPCR. In some embodiments, themodulator or the pharmaceutical agent is an inverse agonist of the GPCR.In some embodiments, the modulator or the pharmaceutical agent is anantagonist of the GPCR. In some embodiments, the inverse agonist orantagonist of the GPCR is an inverse agonist or antagonist of amammalian GPR50. In some embodiments, the mammalian GPR50 is a humanGPR50.

In some embodiments, the modulator or the pharmaceutical agent is asmall molecule.

In some embodiments, the modulator or the pharmaceutical agent is apolypeptide. In some embodiments, the modulator or the pharmaceuticalagent is not an antibody or an antigen-binding fragment thereof. In someembodiments, the modulator or the pharmaceutical agent is a polypeptide,provided that the polypeptide is not an antibody or an antigen-bindingfragment thereof. In some embodiments, the modulator or thepharmaceutical agent is an antibody or an antigen-binding fragmentthereof. In some embodiments, the modulator or the pharmaceutical agentis a lipid. In some embodiments, the modulator or the pharmaceuticalagent is not a polypeptide. In some embodiments, the modulator or thepharmaceutical agent is not a lipid. In some embodiments, the modulatoror the pharmaceutical agent is non-endogenous. In some embodiments, themodulator or the pharmaceutical agent is not endogenous. In someembodiments, the modulator or the pharmaceutical agent is not materialthat a prokaryote or eukaryote naturally produces. In some embodiments,the modulator or the pharmaceutical agent is not material that aprokaryote naturally produces. In some embodiments, the modulator or thepharmaceutical agent is not material that a eukaryote naturallyproduces. In some embodiments, the modulator or the pharmaceutical agentis not material that a mammal naturally produces.

In some embodiments, the modulator or the pharmaceutical agent is aninverse agonist or antagonist with an IC₅₀ of less than about 10 μM, ofless than about 1 μM, of less than about 100 nM, or of less than about10 nM at human, mouse or rat GPR50, preferably at human GPR50. In someembodiments, the modulator or the pharmaceutical agent is an inverseagonist or antagonist with an IC₅₀ of less than a value selected fromthe interval of about 10 nM to 10 μM. In some embodiments, the modulatoror the pharmaceutical agent is an inverse agonist or antagonist with anIC₅₀ of less than a value selected from the interval of about 10 nM to 1μM. In some embodiments, the modulator or the pharmaceutical agent is aninverse agonist or antagonist with an IC₅₀ of less than a value selectedfrom the interval of about 10 nM to 100 nM. In some embodiments, themodulator or the pharmaceutical agent is an inverse agonist orantagonist with an IC₅₀ of less than about 10 μM, of less than about 1μM, of less than about 100 nM, or of less than about 10 nM in GTPγSbinding assay carried out with membrane from transfected CHO cells, orin pigment aggregation assay carried out in transfected melanophores, orin cAMP assay carried out in transfected 293 cells optionallyco-transfected with TSHR, wherein the transfected CHO cells or thetransfected melanophore cells or the transfected 293 cells express arecombinant GPR50 having an amino acid sequence selected from SEQ ID NO:2, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8. In some embodiments, therecombinant GPR50 has the amino acid sequence of SEQ ID NO: 2. In someembodiments, the recombinant GPR50 has the amino acid sequence of SEQ IDNO: 4. In some embodiments, the modulator or the pharmaceutical agent isan inverse agonist or antagonist with an IC₅₀ of less than about 10 μM,of less than about 1 μM, of less than about 100 nM, or of less thanabout 10 nM in said assay. In some embodiments, the modulator or thepharmaceutical agent is an inverse agonist or antagonist with an IC₅₀ insaid assay of less than a value selected from the interval of about 10nM to 10 μM. In some embodiments, the modulator or the pharmaceuticalagent is an inverse agonist or antagonist with an IC₅₀ in said assay ofless than a value selected from the interval of about 10 nM to 1 μM. Insome embodiments, the modulator or the pharmaceutical agent is aninverse agonist or antagonist with an IC₅₀ in said assay of less than avalue selected from the interval of about 10 nM to 100 nM.

In some embodiments, the modulator or the pharmaceutical agent is orallyactive.

In a third aspect, the invention features a pharmaceutical compositioncomprising a modulator of a mammalian GPR50 and a pharmaceuticallyacceptable carrier.

In some embodiments, the modulator of the mammalian GPR50 is a modulatorof a human GPR50. In some embodiments, the modulator of the human GPR50is a modulator of human GPR50 having the amino acid sequence of SEQ IDNO: 2 or SEQ ID NO: 4. In some embodiments, the modulator of the humanGPR50 is a modulator of human GPR50 having the amino acid sequence ofSEQ ID NO: 2. In some embodiments, the modulator of the human GPR50 is amodulator of human GPR50 having the amino acid sequence of SEQ ID NO: 4.

In some embodiments, the modulator is according to the second aspect.

In some embodiments, the modulator of the mammalian GPR50 is an agonist,partial agonist, inverse agonist, or antagonist of the mammalian GPR50.

In some embodiments, the modulator of the mammalian GPR50 is an inverseagonist or antagonist. In some embodiments, the modulator of themammalian GPR50 is an inverse agonist. In some embodiments, themodulator of the mammalian GPR50 is an antagonist.

In some embodiments, the modulator is a small molecule.

In some embodiments, the modulator is a polypeptide. In someembodiments, the modulator is not an antibody or an antigen-bindingfragment thereof. In some embodiments, the modulator is a polypeptide,provided that the polypeptide is not an antibody or an antigen-bindingfragment thereof. In some embodiments, the modulator is an antibody oran antigen-binding fragment thereof. In some embodiments, the modulatoris a lipid. In some embodiments, the modulator is not a polypeptide. Insome embodiments, the modulator is not a lipid. In some embodiments, themodulator is non-endogenous. In some embodiments, the modulator is notendogenous. In some embodiments, the modulator is not material that aprokaryote or eukaryote naturally produces. In some embodiments, themodulator is not material that a prokaryote naturally produces. In someembodiments, the modulator is not material that a eukaryote naturallyproduces. In some embodiments, the modulator is not material that amammal naturally produces.

In some embodiments, the modulator is an inverse agonist or antagonistwith an IC₅₀ of less than about 10 μM, of less than about 1 μM, of lessthan about 100 nM, or of less than about 10 nM at human, mouse or ratGPR50, preferably at human GPR50. In some embodiments, the modulator isan inverse agonist or antagonist with an IC₅₀ of less than a valueselected from the interval of about 10 nM to 10 μM. In some embodiments,the modulator is an inverse agonist or antagonist with an IC₅₀ of lessthan a value selected from the interval of about 10 nM to 1 μM. In someembodiments, the modulator is an inverse agonist or antagonist with anIC₅₀ of less than a value selected from the interval of about 10 nM to100 nM. In some embodiments, the modulator is an inverse agonist orantagonist with an IC₅₀ of less than about 10 μM, of less than about 1μM, of less than about 100 nM, or of less than about 10 nM in GTPγSbinding assay carried out with membrane from transfected CHO cells, orin pigment aggregation assay carried out in transfected melanophores, orin cAMP assay carried out in transfected 293 cells optionallyco-transfected with TSHR, wherein the transfected CHO cells or thetransfected melanophore cells or the transfected 293 cells express arecombinant GPR50 having an amino acid sequence selected from SEQ ID NO:2, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8. In some embodiments, therecombinant GPR50 has the amino acid sequence of SEQ ID NO: 2. In someembodiments, the recombinant GPR50 has the amino acid sequence of SEQ IDNO: 4. In some embodiments, the modulator is an inverse agonist orantagonist with an IC₅₀ of less than about 10 μM, of less than about 1μM, of less than about 100 nM, or of less than about 10 nM in saidassay. In some embodiments, the modulator is an inverse agonist orantagonist with an IC₅₀ in said assay of less than a value selected fromthe interval of about 10 nM to 10 μM. In some embodiments, the modulatoris an inverse agonist or antagonist with an IC₅₀ in said assay of lessthan a value selected from the interval of about 10 nM to 1 μM. In someembodiments, the modulator is an inverse agonist or antagonist with anIC₅₀ in said assay of less than a value selected from the interval ofabout 10 nM to 100 nM.

In some embodiments, the modulator is orally active.

In a fourth aspect, the invention features a method of preparing apharmaceutical composition comprising admixing a modulator of amammalian GPR50 and a pharmaceutically acceptable carrier.

In some embodiments, the modulator is according to the second aspect.

In some embodiments, the modulator of the mammalian GPR50 is a modulatorof a human GPR50. In some embodiments, the modulator of the human GPR50is a modulator of human GPR50 having the amino acid sequence of SEQ IDNO: 2 or SEQ ID NO: 4. In some embodiments, the modulator of the humanGPR50 is a modulator of human GPR50 having the amino acid sequence ofSEQ ID NO: 2. In some embodiments, the modulator of the human GPR50 is amodulator of human GPR50 having the amino acid sequence of SEQ ID NO: 4.

In some embodiments, the modulator of the mammalian GPR50 is an agonist,partial agonist, inverse agonist, or antagonist of the mammalian GPR50.

In some embodiments, the modulator of the mammalian GPR50 is an inverseagonist or antagonist. In some embodiments, the modulator of themammalian GPR50 is an inverse agonist. In some embodiments, themodulator of the mammalian GPR50 is an antagonist.

In some embodiments, the modulator is a small molecule.

In some embodiments, the modulator is a polypeptide. In someembodiments, the modulator is not an antibody or an antigen-bindingfragment thereof. In some embodiments, the modulator is a polypeptide,provided that the polypeptide is not an antibody or an antigen-bindingfragment thereof. In some embodiments, the modulator is an antibody oran antigen-binding fragment thereof. In some embodiments, the modulatoris a lipid. In some embodiments, the modulator is not a polypeptide. Insome embodiments, the modulator is not a lipid. In some embodiments, themodulator is non-endogenous. In some embodiments, the modulator is notendogenous. In some embodiments, the modulator is not material that aprokaryote or eukaryote naturally produces. In some embodiments, themodulator is not material that a prokaryote naturally produces. In someembodiments, the modulator is not material that a eukaryote naturallyproduces. In some embodiments, the modulator is not material that amammal naturally produces.

In some embodiments, the modulator is an inverse agonist or antagonistwith an IC₅₀ of less than about 10 μM, of less than about 1 μM, of lessthan about 100 nM, or of less than about 10 nM at human, mouse or ratGPR50, preferably at human GPR50. In some embodiments, the modulator isan inverse agonist or antagonist with an IC₅₀ of less than a valueselected from the interval of about 10 nM to 10 μM. In some embodiments,the modulator is an inverse agonist or antagonist with an IC₅₀ of lessthan a value selected from the interval of about 10 nM to 1 μM. In someembodiments, the modulator is an inverse agonist or antagonist with anIC₅₀ of less than a value selected from the interval of about 10 nM to100 nM. In some embodiments, the modulator is an inverse agonist orantagonist with an IC₅₀ of less than about 10 μM, of less than about 1μM, of less than about 100 nM, or of less than about 10 nM in GTPγSbinding assay carried out with membrane from transfected CHO cells, orin pigment aggregation assay carried out in transfected melanophores, orin cAMP assay carried out in transfected 293 cells optionallyco-transfected with TSHR, wherein the transfected CHO cells or thetransfected melanophore cells or the transfected 293 cells express arecombinant GPR50 having an amino acid sequence selected from SEQ ID NO:2, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8. In some embodiments, therecombinant GPR50 has the amino acid sequence of SEQ ID NO: 2. In someembodiments, the recombinant GPR50 has the amino acid sequence of SEQ IDNO: 4. In some embodiments, the modulator is an inverse agonist orantagonist with an IC₅₀ of less than about 10 μM, of less than about 1μM, of less than about 100 nM, or of less than about 10 nM in saidassay. In some embodiments, the modulator is an inverse agonist orantagonist with an IC₅₀ in said assay of less than a value selected fromthe interval of about 10 nM to 10 μM. In some embodiments, the modulatoris an inverse agonist or antagonist with an IC₅₀ in said assay of lessthan a value selected from the interval of about 10 nM to 1 μM. In someembodiments, the modulator is an inverse agonist or antagonist with anIC₅₀ in said assay of less than a value selected from the interval ofabout 10 nM to 100 nM.

In some embodiments, the modulator is orally active.

In a fifth aspect, the invention features a method of decreasing bodymass or of decreasing adiposity or of decreasing percentage body fatcomprising administering to a mammal in need thereof a therapeuticallyeffective amount of a modulator of the mammalian GPR50 or apharmaceutical composition comprising the modulator and apharmaceutically acceptable carrier.

In some embodiments, the method is a method of decreasing body mass.

In some embodiments, the method is a method of decreasing adiposity.

In some embodiments, the method is a method of decreasing percentagebody fat.

In some embodiments, the mammal is overweight or obese. In someembodiments, the mammal is overweight. In some embodiments, the mammalis obese.

In some embodiments, the modulator of the mammalian GPR50 is a modulatorof a human GPR50. In some embodiments, the modulator of the human GPR50is a modulator of human GPR50 having the amino acid sequence of SEQ IDNO: 2 or SEQ ID NO: 4. In some embodiments, the modulator of the humanGPR50 is a modulator of human GPR50 having the amino acid sequence ofSEQ ID NO: 2. In some embodiments, the modulator of the human GPR50 is amodulator of human GPR50 having the amino acid sequence of SEQ ID NO: 4.

In some embodiments, the modulator is according to the second aspect.

In some embodiments, the modulator is an agonist, partial agonist,inverse agonist, or antagonist of the mammalian GPR50.

In some embodiments, the modulator is an inverse agonist or antagonistof the mammalian GPR50. In some embodiments, the modulator is an inverseagonist. In some embodiments, the modulator is an antagonist.

In some embodiments, the modulator is a small molecule.

In some embodiments, the modulator is a polypeptide. In someembodiments, the modulator is not an antibody or an antigen-bindingfragment thereof. In some embodiments, the modulator is a polypeptide,provided that the polypeptide is not an antibody or an antigen-bindingfragment thereof. In some embodiments, the modulator is an antibody oran antigen-binding fragment thereof. In some embodiments, the modulatoris a lipid. In some embodiments, the modulator is not a polypeptide. Insome embodiments, the modulator is not a lipid. In some embodiments, themodulator is non-endogenous. In some embodiments, the modulator is notendogenous. In some embodiments, the modulator is not material that aprokaryote or eukaryote naturally produces. In some embodiments, themodulator is not material that a prokaryote naturally produces. In someembodiments, the modulator is not material that a eukaryote naturallyproduces. In some embodiments, the modulator is not material that amammal naturally produces.

In some embodiments, the mammal is a mouse, a rat, or a human. In someembodiments, the mammal is a human.

In some embodiments, the modulator is an inverse agonist or antagonistwith an IC₅₀ of less than about 10 μM, of less than about 1 μM, of lessthan about 100 nM, or of less than about 10 nM at human, mouse or ratGPR50, preferably at human GPR50. In some embodiments, the modulator isan inverse agonist or antagonist with an IC₅₀ of less than a valueselected from the interval of about 10 nM to 10 μM. In some embodiments,the modulator is an inverse agonist or antagonist with an IC₅₀ of lessthan a value selected from the interval of about 10 nM to 1 μM. In someembodiments, the modulator is an inverse agonist or antagonist with anIC₅₀ of less than a value selected from the interval of about 10 nM to100 nM. In some embodiments, the modulator is an inverse agonist orantagonist with an IC₅₀ of less than about 10 μM, of less than about 1μM, of less than about 100 nM, or of less than about 10 nM in GTPγSbinding assay carried out with membrane from transfected CHO cells, orin pigment aggregation assay carried out in transfected melanophores, orin cAMP assay carried out in transfected 293 cells optionallyco-transfected with TSHR, wherein the transfected CHO cells or thetransfected melanophore cells or the transfected 293 cells express arecombinant GPR50 having an amino acid sequence selected from SEQ ID NO:2, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8. In some embodiments, therecombinant GPR50 has the amino acid sequence of SEQ ID NO: 2. In someembodiments, the recombinant GPR50 has the amino acid sequence of SEQ IDNO: 4. In some embodiments, the modulator is an inverse agonist orantagonist with an IC₅₀ of less than about 10 μM, of less than about 1μM, of less than about 100 nM, or of less than about 10 nM in saidassay. In some embodiments, the modulator is an inverse agonist orantagonist with an IC₅₀ in said assay of less than a value selected fromthe interval of about 10 nM to 10 μM. In some embodiments, the modulatoris an inverse agonist or antagonist with an IC₅₀ in said assay of lessthan a value selected from the interval of about 10 nM to 1 μM. In someembodiments, the modulator is an inverse agonist or antagonist with anIC₅₀ in said assay of less than a value selected from the interval ofabout 10 nM to 100 nM.

In some embodiments, said administering is oral.

In a sixth aspect, the invention features a method of preventing ortreating obesity or a condition related thereto comprising administeringto a mammal in need thereof a therapeutically effective amount of amodulator of the mammalian GPR50 or a pharmaceutical compositioncomprising the modulator and a pharmaceutically acceptable carrier.

In some embodiments, the modulator of the mammalian GPR50 is a modulatorof a human GPR50. In some embodiments, the modulator of the human GPR50is a modulator of human GPR50 having the amino acid sequence of SEQ IDNO: 2 or SEQ ID NO: 4 In some embodiments, the modulator of the humanGPR50 is a modulator of human GPR50 having the amino acid sequence ofSEQ ID NO: 2. In some embodiments, the modulator of the human GPR50 is amodulator of human GPR50 having the amino acid sequence of SEQ ID NO: 4.

In some embodiments, the condition related to obesity is selected fromthe group consisting of hypertension, congestive cardiomyopathy,varicosities, pulmonary embolism, coronary heart disease, stroke,idiopathic intracranial hypertension, meralgia parethetica, dyspnea,obstructive sleep apnea, hypoventilation syndrome, Pickwickian syndrome,asthma, immobility, degenerative osteoarthritis, low back pain, striaedistensae or “stretch marks,” venous stasis of the lower extremities,lymphedema, cellulitis, intertrigo, carbuncles, acanthosis nigricans,skin tags, gastro-esophageal reflux disorder, nonalcoholic fattyliver/steatohepatitis, cholelithiasis, hernias, colon cancer, stressincontinence, obesity-related glomerulopathy, breast and uterine cancer,depression and low self-esteem, impaired quality of life, metabolicsyndrome, insulin resistance, Type 2 diabetes, dyslipidemia,atherosclerosis, hyperandrogenemia in women, polycystic ovariansyndrome, dysmenorrhea, infertility, pregnancy complications, and malehypogonadism. In some embodiments, the condition related to obesity isselected from the group consisting of hypertension, insulin resistance,metabolic syndrome, Type 2 diabetes, dyslipidemia, atherosclerosis,coronary heart disease, and stroke.

In some embodiments, the modulator is according to the second aspect.

In some embodiments, the modulator is an agonist, partial agonist,inverse agonist, or antagonist of the mammalian GPR50.

In some embodiments, the modulator is an inverse agonist or antagonistof the mammalian GPR50. In some embodiments, the modulator is an inverseagonist. In some embodiments, the modulator is an antagonist.

In some embodiments, the modulator is a small molecule.

In some embodiments, the modulator is a polypeptide. In someembodiments, the modulator is not an antibody or an antigen-bindingfragment thereof. In some embodiments, the modulator is a polypeptide,provided that the polypeptide is not an antibody or an antigen-bindingfragment thereof. In some embodiments, the modulator is an antibody oran antigen-binding fragment thereof. In some embodiments, the modulatoris a lipid. In some embodiments, the modulator is not a polypeptide. Insome embodiments, the modulator is not a lipid. In some embodiments, themodulator is non-endogenous. In some embodiments, the modulator is notendogenous. In some embodiments, the modulator is not material that aprokaryote or eukaryote naturally produces. In some embodiments, themodulator is not material that a prokaryote naturally produces. In someembodiments, the modulator is not material that a eukaryote naturallyproduces. In some embodiments, the modulator is not material that amammal naturally produces.

In some embodiments, the mammal is a mouse, a rat, or a human. In someembodiments, the mammal is a human.

In some embodiments, the modulator is an inverse agonist or antagonistwith an IC₅₀ of less than about 10 μM, of less than about 1 μM, of lessthan about 100 nM, or of less than about 10 nM at human, mouse or ratGPR50, preferably at human GPR50. In some embodiments, the modulator isan inverse agonist or antagonist with an IC₅₀ of less than a valueselected from the interval of about 10 nM to 10 μM. In some embodiments,the modulator is an inverse agonist or antagonist with an IC₅₀ of lessthan a value selected from the interval of about 10 nM to 1 μM. In someembodiments, the modulator is an inverse agonist or antagonist with anIC₅₀ of less than a value selected from the interval of about 10 nM to100 nM. In some embodiments, the modulator is an inverse agonist orantagonist with an IC₅₀ of less than about 10 μM, of less than about 1μM, of less than about 100 nM, or of less than about 10 nM in GTPγSbinding assay carried out with membrane from transfected CHO cells, orin pigment aggregation assay carried out in transfected melanophores, orin cAMP assay carried out in transfected 293 cells optionallyco-transfected with TSHR, wherein the transfected CHO cells or thetransfected melanophore cells or the transfected 293 cells express arecombinant GPR50 having an amino acid sequence selected from SEQ ID NO:2, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8. In some embodiments, therecombinant GPR50 has the amino acid sequence of SEQ ID NO: 2. In someembodiments, the recombinant GPR50 has the amino acid sequence of SEQ IDNO: 4. In some embodiments, the modulator is an inverse agonist orantagonist with an IC₅₀ of less than about 10 μM, of less than about 1μM, of less than about 100 nM, or of less than about 10 nM in saidassay. In some embodiments, the modulator is an inverse agonist orantagonist with an IC₅₀ in said assay of less than a value selected fromthe interval of about 10 nM to 10 μM. In some embodiments, the modulatoris an inverse agonist or antagonist with an IC₅₀ in said assay of lessthan a value selected from the interval of about 10 nM to 1 μM. In someembodiments, the modulator is an inverse agonist or antagonist with anIC₅₀ in said assay of less than a value selected from the interval ofabout 10 nM to 100 nM.

In some embodiments, said administering is oral.

In a seventh aspect, the invention features a use of a modulator of amammalian GPR50 in the manufacture of a medicament for decreasing bodymass or for decreasing adiposity or for decreasing percentage body fatin the mammal.

In some embodiments, the medicament is for decreasing body mass in themammal.

In some embodiments, the medicament is for decreasing adiposity in themammal.

In some embodiments, the medicament is for decreasing percentage bodyfat in the mammal.

In some embodiments, the mammal is overweight or obese. In someembodiments, the mammal is overweight. In some embodiments, the mammalis obese.

In some embodiments, the modulator of the mammalian GPR50 is a modulatorof a human GPR50. In some embodiments, the modulator of the human GPR50is a modulator of human GPR50 having the amino acid sequence of SEQ IDNO: 2 or SEQ ID NO: 4. In some embodiments, the modulator of the humanGPR50 is a modulator of human GPR50 having the amino acid sequence ofSEQ ID NO: 2. In some embodiments, the modulator of the human GPR50 is amodulator of human GPR50 having the amino acid sequence of SEQ ID NO: 4.

In some embodiments, the modulator is according to the second aspect.

In some embodiments, the modulator is an agonist, partial agonist,inverse agonist, or antagonist of the mammalian GPR50.

In some embodiments, the modulator is an inverse agonist or antagonistof the mammalian GPR50. In some embodiments, the modulator is an inverseagonist. In some embodiments, the modulator is an antagonist.

In some embodiments, the modulator is a small molecule.

In some embodiments, the modulator is a polypeptide. In someembodiments, the modulator is not an antibody or an antigen-bindingfragment thereof. In some embodiments, the modulator is a polypeptide,provided that the polypeptide is not an antibody or an antigen-bindingfragment thereof. In some embodiments, the modulator is an antibody oran antigen-binding fragment thereof. In some embodiments, the modulatoris a lipid. In some embodiments, the modulator is not a polypeptide. Insome embodiments, the modulator is not a lipid. In some embodiments, themodulator is non-endogenous. In some embodiments, the modulator is notendogenous. In some embodiments, the modulator is not material that aprokaryote or eukaryote naturally produces. In some embodiments, themodulator is not material that a prokaryote naturally produces. In someembodiments, the modulator is not material that a eukaryote naturallyproduces. In some embodiments, the modulator is not material that amammal naturally produces.

In some embodiments, the mammal is a mouse, a rat, a non-human primate,or a human. In some embodiments, the mammal is a human.

In some embodiments, the modulator is an inverse agonist or antagonistwith an IC₅₀ of less than about 10 μM, of less than about 1 μM, of lessthan about 100 nM, or of less than about 10 μM at human, mouse or ratGPR50, preferably at human GPR50. In some embodiments, the modulator isan inverse agonist or antagonist with an IC₅₀ of less than a valueselected from the interval of about 10 nM to 10 μM. In some embodiments,the modulator is an inverse agonist or antagonist with an IC₅₀ of lessthan a value selected from the interval of about 10 nM to 1 μM. In someembodiments, the modulator is an inverse agonist or antagonist with anIC₅₀ of less than a value selected from the interval of about 10 nM to100 nM. In some embodiments, the modulator is an inverse agonist orantagonist with an IC₅₀ of less than about 10 μM, of less than about 1μM, of less than about 100 nM, or of less than about 10 nM in GTPγSbinding assay carried out with membrane from transfected CHO cells, orin pigment aggregation assay carried out in transfected melanophores, orin cAMP assay carried out in transfected 293 cells optionallyco-transfected with TSHR, wherein the transfected CHO cells or thetransfected melanophore cells or the transfected 293 cells express arecombinant GPR50 having an amino acid sequence selected from SEQ ID NO:2, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8. In some embodiments, therecombinant GPR50 has the amino acid sequence of SEQ ID NO: 2. In someembodiments, the recombinant GPR50 has the amino acid sequence of SEQ IDNO: 4. In some embodiments, the modulator is an inverse agonist orantagonist with an IC₅₀ of less than about 10 μM, of less than about 1μM, of less than about 100 nM, or of less than about 10 nM in saidassay. In some embodiments, the modulator is an inverse agonist orantagonist with an IC₅₀ in said assay of less than a value selected fromthe interval of about 10 nM to 10 μM. In some embodiments, the modulatoris an inverse agonist or antagonist with an IC₅₀ in said assay of lessthan a value selected from the interval of about 10 nM to 1 μM. In someembodiments, the modulator is an inverse agonist or antagonist with anIC₅₀ in said assay of less than a value selected from the interval ofabout 10 nM to 100 nM.

In some embodiments, the modulator is orally active.

In an eighth aspect, the invention features a use of a modulator of amammalian GPR50 in the manufacture of a medicament for preventing ortreating obesity or a condition related thereto in the mammal.

In some embodiments, the condition related to obesity is selected fromthe group consisting of hypertension, congestive cardiomyopathy,varicosities, pulmonary embolism, coronary heart disease, stroke,idiopathic intracranial hypertension, meralgia parethetica, dyspnea,obstructive sleep apnea, hypoventilation syndrome, Pickwickian syndrome,asthma, immobility, degenerative osteoarthritis, low back pain, striaedistensae or “stretch marks,” venous stasis of the lower extremities,lymphedema, cellulitis, intertrigo, carbuncles, acanthosis nigricans,skin tags, gastro-esophageal reflux disorder, nonalcoholic fattyliver/steatohepatitis, cholelithiasis, hernias, colon cancer, stressincontinence, obesity-related glomerulopathy, breast and uterine cancer,depression and low self-esteem, impaired quality of life, metabolicsyndrome, insulin resistance, Type 2 diabetes, dyslipidemia,atherosclerosis, hyperandrogenemia in women, polycystic ovariansyndrome, dysmenorrhea, infertility, pregnancy complications, and malehypogonadism. In some embodiments, the condition related to obesity isselected from the group consisting of hypertension, insulin resistance,metabolic syndrome, Type 2 diabetes, dyslipidemia, atherosclerosis,coronary heart disease, and stroke.

In some embodiments, the modulator of the mammalian GPR50 is a modulatorof a human GPR50. In some embodiments, the modulator of the human GPR50is a modulator of human GPR50 having the amino acid sequence of SEQ IDNO: 2 or SEQ ID NO: 4. In some embodiments, the modulator of the humanGPR50 is a modulator of human GPR50 having the amino acid sequence ofSEQ ID NO: 2. In some embodiments, the modulator of the human GPR50 is amodulator of human GPR50 having the amino acid sequence of SEQ ID NO: 4.

In some embodiments, the modulator is according to the second aspect.

In some embodiments, the modulator is an agonist, partial agonist,inverse agonist, or antagonist of the mammalian GPR50.

In some embodiments, the modulator is an inverse agonist or antagonistof the mammalian GPR50. In some embodiments, the modulator is an inverseagonist. In some embodiments, the modulator is an antagonist.

In some embodiments, the modulator is a small molecule.

In some embodiments, the modulator is a polypeptide. In someembodiments, the modulator is not an antibody or an antigen-bindingfragment thereof. In some embodiments, the modulator is a polypeptide,provided that the polypeptide is not an antibody or an antigen-bindingfragment thereof. In some embodiments, the modulator is an antibody oran antigen-binding fragment thereof. In some embodiments, the modulatoris a lipid. In some embodiments, the modulator is not a polypeptide. Insome embodiments, the modulator is not a lipid. In some embodiments, themodulator is non-endogenous. In some embodiments, the modulator is notendogenous. In some embodiments, the modulator is not material that aprokaryote or eukaryote naturally produces. In some embodiments, themodulator is not material that a prokaryote naturally produces. In someembodiments, the modulator is not material that a eukaryote naturallyproduces. In some embodiments, the modulator is not material that amammal naturally produces.

In some embodiments, the mammal is a mouse, a rat, a non-human primate,or a human. In some embodiments, the mammal is a human.

In some embodiments, the modulator is an inverse agonist or antagonistwith an IC₅₀ of less than about 10 μM, of less than about 1 μM, of lessthan about 100 nM, or of less than about 10 nM at human, mouse or ratGPR50, preferably at human GPR50. In some embodiments, the modulator isan inverse agonist or antagonist with an IC₅₀ of less than a valueselected from the interval of about 10 nM to 10 μM. In some embodiments,the modulator is an inverse agonist or antagonist with an IC₅₀ of lessthan a value selected from the interval of about 10 nM to 1 μM. In someembodiments, the modulator is an inverse agonist or antagonist with anIC₅₀ of less than a value selected from the interval of about 10 nM to100 nM. In some embodiments, the modulator is an inverse agonist orantagonist with an IC₅₀ of less than about 10 μM, of less than about 1μM, of less than about 100 nM, or of less than about 10 nM in GTPγSbinding assay carried out with membrane from transfected CHO cells, orin pigment aggregation assay carried out in transfected melanophores, orin cAMP assay carried out in transfected 293 cells optionallyco-transfected with TSHR, wherein the transfected CHO cells or thetransfected melanophore cells or the transfected 293 cells express arecombinant GPR50 having an amino acid sequence selected from SEQ ID NO:2, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8. In some embodiments, therecombinant GPR50 has the amino acid sequence of SEQ ID NO: 2. In someembodiments, the recombinant GPR50 has the amino acid sequence of SEQ IDNO: 4. In some embodiments, the modulator is an inverse agonist orantagonist with an IC₅₀ of less than about 10 μM, of less than about 1μM, of less than about 100 nM, or of less than about 10 nM in saidassay. In some embodiments, the modulator is an inverse agonist orantagonist with an IC₅₀ in said assay of less than a value selected fromthe interval of about 10 nM to 10 μM. In some embodiments, the modulatoris an inverse agonist or antagonist with an IC₅₀ in said assay of lessthan a value selected from the interval of about 10 nM to 1 μM. In someembodiments, the modulator is an inverse agonist or antagonist with anIC₅₀ in said assay of less than a value selected from the interval ofabout 10 nM to 100 nM.

In some embodiments, the modulator is orally active.

In a ninth aspect, the invention features a modulator of a mammalianGPR50 for use to decrease body mass in the mammal, for use to decreaseadiposity in the mammal, for use to decrease percentage body fat in themammal, or for use to prevent or treat obesity or a condition relatedthereto in the mammal.

In some embodiments, the condition related to obesity is selected fromthe group consisting of hypertension, congestive cardiomyopathy,varicosities, pulmonary embolism, coronary heart disease, stroke,idiopathic intracranial hypertension, meralgia parethetica, dyspnea,obstructive sleep apnea, hypoventilation syndrome, Pickwickian syndrome,asthma, immobility, degenerative osteoarthritis, low back pain, striaedistensae or “stretch marks,” venous stasis of the lower extremities,lymphedema, cellulitis, intertrigo, carbuncles, acanthosis nigricans,skin tags, gastro-esophageal reflux disorder, nonalcoholic fattyliver/steatohepatitis, cholelithiasis, hernias, colon cancer, stressincontinence, obesity-related glomerulopathy, breast and uterine cancer,depression and low self-esteem, impaired quality of life, metabolicsyndrome, insulin resistance, Type 2 diabetes, dyslipidemia,atherosclerosis, hyperandrogenemia in women, polycystic ovariansyndrome, dysmenorrhea, infertility, pregnancy complications, and malehypogonadism. In some embodiments, the condition related to obesity isselected from the group consisting of hypertension, insulin resistance,metabolic syndrome, Type 2 diabetes, dyslipidemia, atherosclerosis,coronary heart disease, and stroke.

In some embodiments, the modulator of the mammalian GPR50 is a modulatorof a human GPR50. In some embodiments, the modulator of the human GPR50is a modulator of human GPR50 having the amino acid sequence of SEQ IDNO: 2 or SEQ ID NO: 4. In some embodiments, the modulator of the humanGPR50 is a modulator of human GPR50 having the amino acid sequence ofSEQ ID NO: 2. In some embodiments, the modulator of the human GPR50 is amodulator of human GPR50 having the amino acid sequence of SEQ ID NO: 4.

In some embodiments, the modulator is according to the second aspect.

In some embodiments, the modulator is an agonist, partial agonist,inverse agonist, or antagonist of the mammalian GPR50.

In some embodiments, the modulator is an inverse agonist or antagonistof the mammalian GPR50. In some embodiments, the modulator is an inverseagonist. In some embodiments, the modulator is an antagonist.

In some embodiments, the modulator is a small molecule.

In some embodiments, the modulator is a polypeptide. In someembodiments, the modulator is not an antibody or an antigen-bindingfragment thereof. In some embodiments, the modulator is a polypeptide,provided that the polypeptide is not an antibody or an antigen-bindingfragment thereof. In some embodiments, the modulator is an antibody oran antigen-binding fragment thereof. In some embodiments, the modulatoris a lipid. In some embodiments, the modulator is not a polypeptide. Insome embodiments, the modulator is not a lipid. In some embodiments, themodulator is non-endogenous. In some embodiments, the modulator is notendogenous. In some embodiments, the modulator is not material that aprokaryote or eukaryote naturally produces. In some embodiments, themodulator is not material that a prokaryote naturally produces. In someembodiments, the modulator is not material that a eukaryote naturallyproduces. In some embodiments, the modulator is not material that amammal naturally produces.

In some embodiments, the mammal is a mouse, a rat, a non-human primate,or a human. In some embodiments, the mammal is a human.

In some embodiments, the modulator is an inverse agonist or antagonistwith an IC₅₀ of less than about 10 μM, of less than about 1 μM, of lessthan about 100 nM, or of less than about 10 nM at human, mouse or ratGPR50, preferably at human GPR50. In some embodiments, the modulator isan inverse agonist or antagonist with an IC₅₀ of less than a valueselected from the interval of about 10 nM to 10 μM. In some embodiments,the modulator is an inverse agonist or antagonist with an IC₅₀ of lessthan a value selected from the interval of about 10 nM to 1 μM. In someembodiments, the modulator is an inverse agonist or antagonist with anIC₅₀ of less than a value selected from the interval of about 10 nM to100 nM. In some embodiments, the modulator is an inverse agonist orantagonist with an IC₅₀ of less than about 10 μM, of less than about 1μM, of less than about 100 nM, or of less than about 10 nM in GTPγSbinding assay carried out with membrane from transfected CHO cells, orin pigment aggregation assay carried out in transfected melanophores, orin cAMP assay carried out in transfected 293 cells optionallyco-transfected with TSHR, wherein the transfected CHO cells or thetransfected melanophore cells or the transfected 293 cells express arecombinant GPR50 having an amino acid sequence selected from SEQ ID NO:2, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8. In some embodiments, therecombinant GPR50 has the amino acid sequence of SEQ ID NO: 2. In someembodiments, the recombinant GPR50 has the amino acid sequence of SEQ IDNO: 4. In some embodiments, the modulator is an inverse agonist orantagonist with an IC₅₀ of less than about 10 μM, of less than about 1μM, of less than about 100 nM, or of less than about 10 nM in saidassay. In some embodiments, the modulator is an inverse agonist orantagonist with an IC₅₀ in said assay of less than a value selected fromthe interval of about 10 nM to 10 μM. In some embodiments, the modulatoris an inverse agonist or antagonist with an IC₅₀ in said assay of lessthan a value selected from the interval of about 10 nM to 1 μM. In someembodiments, the modulator is an inverse agonist or antagonist with anIC₅₀ in said assay of less than a value selected from the interval ofabout 10 nM to 100 nM.

In some embodiments, the modulator is orally active.

In a tenth aspect, the invention features a pharmaceutical compositioncomprising a modulator of a mammalian GPR50 and a pharmaceuticallyacceptable carrier for use to decrease body mass in the mammal, for useto decrease adiposity in the mammal, for use to decrease percentage bodyfat in the mammal, or for use to prevent or treat obesity or a conditionrelated thereto in the mammal.

In some embodiments, the condition related to obesity is selected fromthe group consisting of hypertension, congestive cardiomyopathy,varicosities, pulmonary embolism, coronary heart disease, stroke,idiopathic intracranial hypertension, meralgia parethetica, dyspnea,obstructive sleep apnea, hypoventilation syndrome, Pickwickian syndrome,asthma, immobility, degenerative osteoarthritis, low back pain, striaedistensae or “stretch marks,” venous stasis of the lower extremities,lymphedema, cellulitis, intertrigo, carbuncles, acanthosis nigricans,skin tags, gastro-esophageal reflux disorder, nonalcoholic fattyliver/steatohepatitis, cholelithiasis, hernias, colon cancer, stressincontinence, obesity-related glomerulopathy, breast and uterine cancer,depression and low self-esteem, impaired quality of life, metabolicsyndrome, insulin resistance, Type 2 diabetes, dyslipidemia,atherosclerosis, hyperandrogenemia in women, polycystic ovariansyndrome, dysmenorrhea, infertility, pregnancy complications, and malehypogonadism. In some embodiments, the condition related to obesity isselected from the group consisting of hypertension, insulin resistance,metabolic syndrome, Type 2 diabetes, dyslipidemia, atherosclerosis,coronary heart disease, and stroke.

In some embodiments, the modulator of the mammalian GPR50 is a modulatorof a human GPR50. In some embodiments, the modulator of the human GPR50is a modulator of human GPR50 having the amino acid sequence of SEQ IDNO: 2 or SEQ ID NO: 4. In some embodiments, the modulator of the humanGPR50 is a modulator of human GPR50 having the amino acid sequence ofSEQ ID NO: 2. In some embodiments, the modulator of the human GPR50 is amodulator of human GPR50 having the amino acid sequence of SEQ ID NO: 4.

In some embodiments, the modulator is according to the second aspect.

In some embodiments, the modulator is an agonist, partial agonist,inverse agonist, or antagonist of the mammalian GPR50.

In some embodiments, the modulator is an inverse agonist or antagonistof the mammalian GPR50. In some embodiments, the modulator is an inverseagonist. In some embodiments, the modulator is an antagonist.

In some embodiments, the modulator is a small molecule.

In some embodiments, the modulator is a polypeptide. In someembodiments, the modulator is not an antibody or an antigen-bindingfragment thereof. In some embodiments, the modulator is a polypeptide,provided that the polypeptide is not an antibody or an antigen-bindingfragment thereof. In some embodiments, the modulator is an antibody oran antigen-binding fragment thereof. In some embodiments, the modulatoris a lipid. In some embodiments, the modulator is not a polypeptide. Insome embodiments, the modulator is not a lipid. In some embodiments, themodulator is non-endogenous. In some embodiments, the modulator is notendogenous. In some embodiments, the modulator is not material that aprokaryote or eukaryote naturally produces. In some embodiments, themodulator is not material that a prokaryote naturally produces. In someembodiments, the modulator is not material that a eukaryote naturallyproduces. In some embodiments, the modulator is not material that amammal naturally produces.

In some embodiments, the mammal is a mouse, a rat, a non-human primate,or a human. In some embodiments, the mammal is a human.

In some embodiments, the modulator is an inverse agonist or antagonistwith an IC₅₀ of less than about 10 μM, of less than about 1 μM, of lessthan about 100 nM, or of less than about 10 nM at human, mouse or ratGPR50, preferably at human GPR50. In some embodiments, the modulator isan inverse agonist or antagonist with an IC₅₀ of less than a valueselected from the interval of about 10 nM to 10 μM. In some embodiments,the modulator is an inverse agonist or antagonist with an IC₅₀ of lessthan a value selected from the interval of about 10 nM to 1 μM. In someembodiments, the modulator is an inverse agonist or antagonist with anIC₅₀ of less than a value selected from the interval of about 10 nM to100 nM. In some embodiments, the modulator is an inverse agonist orantagonist with an IC₅₀ of less than about 10 μM, of less than about 1μM, of less than about 100 nM, or of less than about 10 nM in GTPγSbinding assay carried out with membrane from transfected CHO cells, orin pigment aggregation assay carried out in transfected melanophores, orin cAMP assay carried out in transfected 293 cells optionallyco-transfected with TSHR, wherein the transfected CHO cells or thetransfected melanophore cells or the transfected 293 cells express arecombinant GPR50 having an amino acid sequence selected from SEQ ID NO:2, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8. In some embodiments, therecombinant GPR50 has the amino acid sequence of SEQ ID NO: 2. In someembodiments, the recombinant GPR50 has the amino acid sequence of SEQ IDNO: 4. In some embodiments, the modulator is an inverse agonist orantagonist with an IC₅₀ of less than about 10 μM, of less than about 1μM, of less than about 100 nM, or of less than about 10 nM in saidassay. In some embodiments, the modulator is an inverse agonist orantagonist with an IC₅₀ in said assay of less than a value selected fromthe interval of about 10 nM to 10 μM. In some embodiments, the modulatoris an inverse agonist or antagonist with an IC₅₀ in said assay of lessthan a value selected from the interval of about 10 μM to 1 μM. In someembodiments, the modulator is an inverse agonist or antagonist with anIC₅₀ in said assay of less than a value selected from the interval ofabout 10 nM to 100 nM.

In some embodiments, the modulator is orally active.

In an eleventh aspect, the invention features a method of identifying acandidate compound as a ligand of a GPCR comprising an amino acidsequence selected from the group consisting of:

-   -   (a) the amino acid sequence of SEQ ID NO: 2;    -   (b) amino acids 2-617 of SEQ ID NO: 2;    -   (c) amino acids 2-617 of SEQ ID NO:2, wherein the GPCR does not        comprise amino acids 1-617 of SEQ ID NO: 2;    -   (d) the amino acid sequence of (a), (b) or (c), wherein SEQ ID        NO: 2 comprises any combination of a substitution of serine at        amino acid position 493 of SEQ ID NO: 2 with asparagine, a        substitution of threonine at amino acid position 532 of SEQ ID        NO: 2 with alanine, and a substitution of valine at amino acid        position 606 of SEQ ID NO: 2 with isoleucine;    -   (c) the amino acid sequence of SEQ ID NO: 4;    -   (d) amino acids 2-613 of SEQ ID NO: 4;    -   (e) amino acids 2-613 of SEQ ID NO: 4, wherein the GPCR does not        comprise amino acids 1-613 of SEQ ID NO: 4;    -   (f) the amino acid sequence of (e), (i) or (g), wherein SEQ ID        NO: 4 comprises any combination of a substitution of asparagine        at amino acid position 493 of SEQ ID NO: 4 with serine, a        substitution of alanine at amino acid position 528 of SEQ ID NO:        4 with threonine, and a substitution of valine at amino acid        position 602 of SEQ ID NO: 4 with isoleucine;    -   (g) the amino acid sequence of a G protein-coupled receptor        encoded by a polynucleotide that is amplifiable by polymerase        chain reaction (PCR) on a human DNA sample using specific        primers SEQ ID NO: 9 and SEQ ID NO: 10;    -   (h) the amino acid sequence of a G protein-coupled receptor        encoded by a polynucleotide hybridizing at high stringency to        the complement of SEQ ID NO: 1 or SEQ ID NO: 3;    -   (i) the amino acid sequence of a G protein-coupled receptor        having at least about 75%, at least about 80%, at least about        85%, at least about 90% or at least about 95% identity to SEQ ID        NO: 2 or SEQ ID NO: 4;    -   (j) the amino acid sequence of SEQ ID NO: 6;    -   (k) amino acids 2-591 of SEQ ID NO: 6;    -   (l) amino acids 2-591 of SEQ ID NO: 6 wherein the GPCR does not        comprise amino acids 1-591 of SEQ ID NO: 6;    -   (m) the amino acid sequence of a G protein-coupled receptor        encoded by a polynucleotide hybridizing at high stringency to        the complement of SEQ ID NO: 5;    -   (n) the amino acid sequence of a G protein-coupled receptor        having at least about 75%, at least about 80%, at least about        85%, at least about 90% or at least about 95% identity to SEQ ID        NO: 6;    -   (o) the amino acid sequence of SEQ ID NO:8;    -   (p) amino acids 2-594 of SEQ ID NO:8;    -   (q) amino acids 2-594 of SEQ ID NO:8, wherein the GPCR does not        comprise amino acids 1-594 of SEQ ID NO:8;    -   (r) the amino acid sequence of a G protein-coupled receptor        encoded by a polynucleotide hybridizing at high stringency to        the complement of SEQ ID NO: 7;    -   (s) the amino acid sequence of a G protein-coupled receptor        having at least about 75%, at least about 80%, at least about        85%, at least about 90% or at least about 95% identity to SEQ ID        NO: 8; and    -   (t) the amino acid sequence of a G protein-coupled receptor that        is a constitutively active version of a receptor having SEQ ID        NO: 2 or SEQ ID NO: 4;    -   or a variant or biologically active fragment thereof;        comprising the steps of:    -   (a′) contacting said GPCR with an optionally labeled known        ligand to the GPCR in the presence or absence of the candidate        compound;    -   (b′) detecting the complex between the known ligand and said        GPCR; and    -   (c′) determining whether less of said complex is formed in the        presence of the candidate compound than in the absence of the        candidate compound;        wherein said determination is indicative of the candidate        compound being a ligand of said receptor.

In some embodiments, the GPCR comprises the amino acid sequence of a Gprotein-coupled receptor having at least about 75%, at least about 80%,at least about 85%, at least about 90% or at least about 95% identity toSEQ ID NO: 2 or SEQ ID NO: 4.

In some embodiments, the G protein-coupled receptor having at leastabout 75%, at least about 80%, at least about 85%, at least about 90% orat least about 95% identity to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6or SEQ ID NO: 8 is an endogenous GPCR. In some embodiments, the Gprotein-coupled receptor having at least about 75%, at least about 80%,at least about 85%, at least about 90% or at least about 95% identity toSEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8 is a mammalianendogenous GPCR. In some embodiments, the G protein-coupled receptorhaving at least about 75%, at least about 80%, at least about 85%, atleast about 90% or at least about 95% identity to SEQ ID NO: 2, SEQ IDNO: 4, SEQ ID NO: 6 or SEQ ID NO: 8 is a non-endogenous GPCR.

In some embodiments, the G protein-coupled receptor that is aconstitutively active version of a receptor having SEQ ID NO: 2 or SEQID NO: 4 is an endogenous G protein-coupled receptor. In someembodiments, the G protein-coupled receptor that is a constitutivelyactive version of a receptor having SEQ ID NO: 2 or SEQ ID NO: 4 is anon-endogenous G protein-coupled receptor.

In some embodiments, PCR is RT-PCR.

In some embodiments, the human DNA is human cDNA derived from a tissueor cell type that expresses GPR50. In some embodiments, the human EDNAis derived from hypothalamus or pituitary.

In some embodiments, the G protein-coupled receptor encoded by apolynucleotide that is amplifiable by polymerase chain reaction (PCR) ona human DNA sample using specific primers SEQ ID NO: 9 and SEQ ID NO: 10is an endogenous GPR50 G protein-coupled receptor.

In some embodiments, the GPCR is recombinant.

In some embodiments, the GPCR is endogenous. In some embodiments, theGPCR that is endogenous is a mammalian endogenous GPCR. In someembodiments, the mammalian endogenous GPCR is a mammalian endogenousGPR50. In some embodiments, the GPCR is non-endogenous.

In some embodiments, the GPCR is a mammalian GPR50.

In some embodiments, the G protein-coupled receptor encoded by apolynucleotide hybridizing at high stringency to the complement of SEQID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7 is an endogenousGPCR. In some embodiments, the G protein-coupled receptor encoded by apolynucleotide hybridizing at high stringency to the complement of SEQID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7 that is anendogenous GPCR is a mammalian GPCR. In some embodiments, the Gprotein-coupled receptor encoded by a polynucleotide hybridizing at highstringency to the complement of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5or SEQ ID NO: 7 exhibits a detectable level of constitutive activity. Insome embodiments, the constitutive activity is for lowering a level ofintracellular cAMP. In some embodiments, the constitutive activity isfor causing melanophore cells to undergo pigment aggregation. In certainembodiments, the G protein-coupled receptor encoded by a polynucleotidehybridizing at high stringency to the complement of SEQ ID NO: 1, SEQ IDNO: 3, SEQ ID NO: 5 or SEQ ID NO: 7 specifically binds an antibody thatrecognizes an mammalian endogenous GPR50 (an antibody that recognizes anendogenous mammalian GPR50 can be obtained commercially from, e.g.,Advanced Targeting Systems, San Diego, Calif.; and CHEMICONInternational, Inc., Temecula, Calif.) or specifically binds a knownligand of an mammalian endogenous GPR50. In certain embodiments, theknown ligand of the mammalian endogenous GPR50 is an endogenous ligandof the mammalian endogenous GPR50.

In some embodiments, the known ligand to the GPCR is a known ligand to amammalian GPR50. In some embodiments, the known ligand to the GPCR is aknown ligand to a human GPR50.

In some embodiments, the known ligand to the GPCR is a known ligand toSEQ ID NO: 2. In some embodiments, the known ligand to the GPCR is aknown ligand to SEQ ID NO: 4. In some embodiments, the known ligand tothe GPCR is a known ligand to SEQ ID NO: 6. In some embodiments, theknown ligand to the GPCR is a known ligand to SEQ ID NO: 8.

In some embodiments, the known ligand is not an antibody or anantigen-binding fragment thereof.

In some embodiments, the known ligand is radiolabeled.

In some embodiments, the candidate compound is a small molecule.

In some embodiments, the candidate compound is a polypeptide. In someembodiments, the candidate compound is not an antibody or anantigen-binding fragment thereof. In some embodiments, the candidatecompound is a polypeptide, provided that the polypeptide is not anantibody or an antigen-binding fragment thereof. In some embodiments,the candidate compound is an antibody or an antigen-binding fragmentthereof. In some embodiments, the candidate compound is a lipid. In someembodiments, the candidate compound is not a polypeptide. In someembodiments, the candidate compound is not a lipid. In some embodiments,the candidate compound is non-endogenous. In some embodiments, thecandidate compound is not endogenous. In some embodiments, the candidatecompound is not material that a prokaryote or eukaryote naturallyproduces. In some embodiments, the candidate compound is not materialthat a prokaryote naturally produces. In some embodiments, the candidatecompound is not material that a eukaryote naturally produces. In someembodiments, the candidate compound is not material that a mammalnaturally produces. In some embodiments, the candidate compound is acompound not known to be a ligand of the GPCR.

In some embodiments, the method is for screening candidate compounds asmodulators of body mass or of adiposity or of percentage body fat in asubject or as pharmaceutical agents for obesity or a condition relatedthereto. In some embodiments, the condition related to obesity isselected from the group consisting of hypertension, congestivecardiomyopathy, varicosities, pulmonary embolism, coronary heartdisease, stroke, idiopathic intracranial hypertension, meralgiaparethetica, dyspnea, obstructive sleep apnea, hypoventilation syndrome,Pickwickian syndrome, asthma, immobility, degenerative osteoarthritis,low back pain, striae distensae or “stretch marks,” venous stasis of thelower extremities, lymphedema, cellulitis, intertrigo, carbuncles,acanthosis nigricans, skin tags, gastro-esophageal reflux disorder,nonalcoholic fatty liver/steatohepatitis, cholelithiasis, hernias, coloncancer, stress incontinence, obesity-related glomerulopathy, breast anduterine cancer, depression and low self-esteem, impaired quality oflife, metabolic syndrome, insulin resistance, Type 2 diabetes,dyslipidemia, atherosclerosis, hyperandrogenemia in women, polycysticovarian syndrome, dysmenorrhea, infertility, pregnancy complications,and male hypogonadism. In some embodiments, the condition related toobesity is selected from the group consisting of hypertension, insulinresistance, metabolic syndrome, Type 2 diabetes, dyslipidemia,atherosclerosis, coronary heart disease, and stroke. In someembodiments, the subject is a mammal. In some embodiments, the subjectis a mammal selected from the group consisting of mouse, rat and human.In some embodiments, the subject is a human.

In some embodiments, the method is for screening candidate compounds ascompounds useful in radio-imaging for identifying a subject at risk forobesity or a condition related thereto. In some embodiments, the subjectis a human.

In a twelfth aspect, the invention features a transgenic non-humanmammal comprising a disruption in an endogenous GPR50 gene, wherein thedisruption is homozygous, the transgenic non-human mammal lacksproduction of functional GPR50 protein and exhibits, relative to thewild-type mammal, a decreased weight gain induced by a high fat diet.

In some embodiments, the transgenic non-human mammal is a mouse, a rator a pig. In some embodiments, the transgenic non-human mammal is amouse.

In an thirteenth aspect, the invention features a method of identifyinga candidate compound as a modulator of body mass or of adiposity or ofpercentage body fat in a subject, the method comprising:

-   providing a transgenic non-human mammal according to the twelfth    aspect;-   administering the candidate compound to the transgenic non-human    mammal; and-   determining whether the decreased weight gain induced by a high-fat    diet is modulated by the candidate compound, thereby identifying the    candidate compound as a modulator of body mass or of adiposity or of    percentage body fat in the subject.

In some embodiments, the modulator of body mass or of adiposity or ofpercentage body fat in a subject is a modulator of body mass in thesubject.

In some embodiments, the modulator of body mass or of adiposity or ofpercentage body fat in a subject is a modulator of adiposity in thesubject.

In some embodiments, the modulator of body mass or of adiposity or ofpercentage body fat in a subject is a modulator of percentage body fatin the subject.

In some embodiments, the transgenic non-human mammal is a mouse, a rator a pig. In some embodiments, the transgenic non-human mammal is amouse.

In some embodiments, the candidate compound is a small molecule.

In some embodiments, the candidate compound is a polypeptide. In someembodiments, the candidate compound is not an antibody or anantigen-binding fragment thereof. In some embodiments, the candidatecompound is a polypeptide, provided that the polypeptide is not anantibody or an antigen-binding fragment thereof. In some embodiments,the candidate compound is an antibody or an antigen-binding fragmentthereof. In some embodiments, the candidate compound is a lipid. In someembodiments, the candidate compound is not a polypeptide. In someembodiments, the candidate compound is not a lipid. In some embodiments,the candidate compound is non-endogenous. In some embodiments, thecandidate compound is not endogenous. In some embodiments, the candidatecompound is not material that a prokaryote or eukaryote naturallyproduces. In some embodiments, the candidate compound is not materialthat a prokaryote naturally produces. In some embodiments, the candidatecompound is not material that a eukaryote naturally produces. In someembodiments, the candidate compound is not material that a mammalnaturally produces.

In some embodiments, the subject is a mammal. In some embodiments, thesubject that is a mammal is selected from the group consisting of mouse,rat and human. In some embodiments, the subject that is a mammal is ahuman.

In a fourteenth aspect, the invention features use of a GPCR to screencandidate compounds as modulators of body mass or of adiposity or ofpercentage body fat in a mammal or as pharmaceutical agents for obesityor a condition related thereto, wherein the GPCR comprises an amino acidsequence selected from the group consisting of:

-   -   (a) the amino acid sequence of SEQ ID NO: 2;    -   (b) amino acids 2-617 of SEQ ID NO: 2;    -   (c) amino acids 2-617 of SEQ ID NO:2, wherein the GPCR does not        comprise amino acids 1-617 of SEQ ID NO: 2;    -   (d) the amino acid sequence of (a), (b) or (c), wherein SEQ ID        NO: 2 comprises any combination of a substitution of serine at        amino acid position 493 of SEQ ID NO: 2 with asparagine, a        substitution of threonine at amino acid position 532 of SEQ ID        NO: 2 with alanine, and a substitution of valine at amino acid        position 606 of SEQ ID NO: 2 with isoleucine;    -   (e) the amino acid sequence of SEQ ID NO: 4;    -   (f) amino acids 2-613 of SEQ ID NO: 4;    -   (g) amino acids 2-613 of SEQ ID NO: 4, wherein the GPCR does not        comprise amino acids 1-613 of SEQ ID NO: 4;    -   (h) the amino acid sequence of (e), (f) or (g), wherein SEQ ID        NO: 4 comprises any combination of a substitution of asparagine        at amino acid position 493 of SEQ ID NO: 4 with serine, a        substitution of alanine at amino acid position 528 of SEQ ID NO:        4 with threonine, and a substitution of valine at amino acid        position 602 of SEQ ID NO: 4 with isoleucine;    -   (i) the amino acid sequence of a G protein-coupled receptor        encoded by a polynucleotide that is amplifiable by polymerase        chain reaction (PCR) on a human DNA sample using specific        primers SEQ ID NO: 9 and SEQ ID NO: 10;    -   (j) the amino acid sequence of a G protein-coupled receptor        encoded by a polynucleotide hybridizing at high stringency to        the complement of SEQ ID NO: 1 or SEQ ID NO: 3;    -   (k) the amino acid sequence of an endogenous G protein-coupled        receptor having at least about 95% identity to SEQ ID NO: 2 or        SEQ ID NO: 4;    -   (l) the amino acid sequence of SEQ ID NO: 6;    -   (m) amino acids 2-591 of SEQ ID NO: 6;    -   (n) amino acids 2-591 of SEQ ID NO: 6 wherein the GPCR does not        comprise amino acids 1-591 of SEQ ID NO: 6;    -   (o) the amino acid sequence of a G protein-coupled receptor        encoded by a polynucleotide hybridizing at high stringency to        the complement of SEQ ID NO: 5;    -   (p) the amino acid sequence of a G protein-coupled receptor        having at least about 75%, at least about 80%, at least about        85%, at least about 90% or at least about 95% identity to SEQ ID        NO: 6;    -   (q) the amino acid sequence of SEQ ID NO:8;    -   (r) amino acids 2-594 of SEQ ID NO:8;    -   (s) amino acids 2-594 of SEQ ID NO:8, wherein the GPCR does not        comprise amino acids 1-594 of SEQ ID NO:8;    -   (t) the amino acid sequence of a G protein-coupled receptor        encoded by a polynucleotide hybridizing at high stringency to        the complement of SEQ ID NO: 7;    -   (u) the amino acid sequence of a G protein-coupled receptor        having at least about 75%, at least about 80%, at least about        85%, at least about 90% or at least about 95% identity to SEQ ID        NO: 8; and    -   (v) the amino acid sequence of a G protein-coupled receptor that        is a constitutively active version of a receptor having SEQ ID        NO: 2 or SEQ ID NO: 4;    -   or a variant or biologically active fragment thereof.

In some embodiments, the GPCR comprises the amino acid sequence of SEQID NO: 2 or SEQ ID NO: 4.

In some embodiments, the GPCR comprises the amino acid sequence of a Gprotein-coupled receptor having at least about 75%, at least about 80%,at least about 85%, at least about 90% or at least about 95% identity toSEQ ID NO: 2 or SEQ ID NO: 4.

In some embodiments, the G protein-coupled receptor having at leastabout 75%, at least about 80%, at least about 85%, at least about 90% orat least about 95% identity to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6or SEQ ID NO: 8 is an endogenous GPCR. In some embodiments, the Gprotein-coupled receptor having at least about 75%, at least about 80%,at least about 85%, at least about 90% or at least about 95% identity toSEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8 is a mammalianendogenous GPCR. In some embodiments, the G protein-coupled receptorhaving at least about 75%, at least about 80%, at least about 85%, atleast about 90% or at least about 95% identity to SEQ ID NO: 2, SEQ IDNO: 4, SEQ ID NO: 6 or SEQ ID NO: 8 is a non-endogenous GPCR.

In some embodiments, the G protein-coupled receptor that is aconstitutively active version of a receptor having SEQ ID NO: 2 or SEQID NO: 4 is an endogenous G protein-coupled receptor. In someembodiments, the G protein-coupled receptor that is a constitutivelyactive version of a receptor having SEQ ID NO: 2 or SEQ ID NO: 4 is anon-endogenous G protein-coupled receptor.

In some embodiments, the modulator of body mass in a mammal is acompound that decreases body mass in a mammal. In some embodiments, themodulator of adiposity in a mammal is a compound that decreasesadiposity in a mammal. In some embodiments, the modulator of percentagebody fat in a mammal is a compound that decreases percentage body fat ina mammal. In some embodiments, the mammal is a human.

In some embodiments, the modulator of body mass in a mammal is acompound that increases body mass in a mammal. In some embodiments, themodulator of adiposity in a mammal is a compound that increasesadiposity in a mammal. In some embodiments, the modulator of percentagebody fat in a mammal is a compound that increases percentage body fat ina mammal. In some embodiments, the mammal is a human. In someembodiments, the compound that increases body mass or increasesadiposity or increases percentage body fat in a mammal is a compound forpreventing or treating cachexia, wasting, AIDS-related weight loss,cancer-related weight loss, anorexia, or bulimia in the mammal. In someembodiments, the compound that increases body mass or increasesadiposity or increases percentage body fat in a mammal is a compound foruse to prevent or treat cachexia, wasting, AIDS-related weight loss,cancer-related weight loss, anorexia, or bulimia in the mammal.

In some embodiments, PCR is RT-PCR.

In some embodiments, the human DNA is human cDNA derived from a tissueor cell type that expresses GPR50. In some embodiments, the human cDNAis derived from hypothalamus or pituitary.

In some embodiments, the G protein-coupled receptor encoded by apolynucleotide that is amplifiable by polymerase chain reaction (PCR) ona human DNA sample using specific primers SEQ ID NO: 9 and SEQ ID NO: 10is an endogenous GPR50 G protein-coupled receptor.

In some embodiments, the G protein-coupled receptor encoded by apolynucleotide hybridizing at high stringency to the complement of SEQID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7 is an endogenousGPCR. In some embodiments, the G protein-coupled receptor encoded by apolynucleotide hybridizing at high stringency to the complement of SEQID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7 that is anendogenous GPCR is a mammalian GPCR. In some embodiments, the Gprotein-coupled receptor encoded by a polynucleotide hybridizing at highstringency to the complement of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5or SEQ ID NO: 7 exhibits a detectable level of constitutive activity. Insome embodiments, the constitutive activity is for lowering a level ofintracellular cAMP. In some embodiments, the constitutive activity isfor causing melanophore cells to undergo pigment aggregation. In certainembodiments, the G protein-coupled receptor encoded by a polynucleotidehybridizing at high stringency to the complement of SEQ ID NO: 1, SEQ IDNO: 3, SEQ ID NO: 5 or SEQ ID NO: 7 specifically binds an antibody thatrecognizes an mammalian endogenous GPR50 (an antibody that recognizes anendogenous mammalian GPR50 can be obtained commercially from, e.g.,Advanced Targeting Systems, San Diego, Calif.; and CHEMICONInternational, Inc., Temecula, Calif.) or specifically binds a knownligand of an mammalian endogenous GPR50. In certain embodiments, theknown ligand of the mammalian endogenous GPR50 is an endogenous ligandof the mammalian endogenous GPR50.

In some embodiments, the GPCR is recombinant.

In some embodiments, the GPCR is endogenous. In some embodiments, theGPCR that is endogenous is a mammalian endogenous GPCR. In someembodiments, the mammalian endogenous GPCR is a mammalian endogenousGPR50. In some embodiments, the GPCR is non-endogenous.

In some embodiments, the GPCR is a mammalian GPR50.

In some embodiments, the candidate compound is a small molecule.

In some embodiments, the candidate compound is a polypeptide. In someembodiments, the candidate compound is not an antibody or anantigen-binding fragment thereof. In some embodiments, the candidatecompound is a polypeptide, provided that the polypeptide is not anantibody or an antigen-binding fragment thereof. In some embodiments,the candidate compound is an antibody or an antigen-binding fragmentthereof. In some embodiments, the candidate compound is a lipid. In someembodiments, the candidate compound is not a polypeptide. In someembodiments, the candidate compound is not a lipid. In some embodiments,the candidate compound is non-endogenous. In some embodiments, thecandidate compound is not endogenous. In some embodiments, the candidatecompound is not material that a prokaryote or eukaryote naturallyproduces. In some embodiments, the candidate compound is not materialthat a prokaryote naturally produces. In some embodiments, the candidatecompound is not material that a eukaryote naturally produces. In someembodiments, the candidate compound is not material that a mammalnaturally produces. In some embodiments, the candidate compound is acompound not known to be a ligand of the GPCR. In some embodiments, thecandidate compound is a compound not known to inhibit or stimulatefunctionality of the GPCR. In some embodiments, the candidate compoundis a compound not known to be an agonist of the GPCR. In someembodiments, the candidate compound is a compound not known to be apartial agonist of the GPCR. In some embodiments, the candidate compoundis a compound not known to be an inverse agonist of the GPCR. In someembodiments, the candidate compound is a compound not known to be anantagonist of the GPCR.

In some embodiments, the condition related to obesity is selected fromthe group consisting of hypertension, congestive cardiomyopathy,varicosities, pulmonary embolism, coronary heart disease, stroke,idiopathic intracranial hypertension, meralgia parethetica, dyspnea,obstructive sleep apnea, hypoventilation syndrome, Pickwickian syndrome,asthma, immobility, degenerative osteoarthritis, low back pain, striaedistensae or “stretch marks,” venous stasis of the lower extremities,lymphedema, cellulitis, intertrigo, carbuncles, acanthosis nigricans,skin tags, gastro-esophageal reflux disorder, nonalcoholic fattyliver/steatohepatitis, cholelithiasis, hernias, colon cancer, stressincontinence, obesity-related glomerulopathy, breast and uterine cancer,depression and low self-esteem, impaired quality of life, metabolicsyndrome, insulin resistance, Type 2 diabetes, dyslipidemia,atherosclerosis, hyperandrogenemia in women, polycystic ovariansyndrome, dysmenorrhea, infertility, pregnancy complications, and malehypogonadism. In some embodiments, the condition related to obesity isselected from the group consisting of hypertension, insulin resistance,metabolic syndrome, Type 2 diabetes, dyslipidemia, atherosclerosis,coronary heart disease, and stroke.

In some embodiments, the screen is for an agonist of the GPCR. In someembodiments, the screen is for a partial agonist of the GPCR. In someembodiments, the screen is for an inverse agonist of the GPCR. In someembodiments, the screen is for an antagonist of the GPCR.

In some embodiments, the mammal is selected from the group consisting ofmouse, rat, non-human primate, and human. In some embodiments, themammal is a human.

Applicant reserves the right to exclude any one or more candidatecompounds from any of the embodiments of the invention. Applicantreserves the right to exclude any one or more modulators from any of theembodiments of the invention. By way of example and not limitation,Applicant reserves the right to exclude any one or more inverse agonistsor antagonists from any of the embodiments of the invention. Applicantreserves the right to exclude any polynucleotide or polypeptide from anyof the embodiments of the invention. Applicant additionally reserves theright to exclude any condition related to obesity from any of theembodiments of the invention. It is also expressly contemplated thatconditions related to obesity of the invention can be included in anembodiment either individually or in any combination. Applicantadditionally reserves the right to exclude any disorder ameliorated byincreasing body mass from any of the embodiments of the invention. It isalso expressly contemplated that disorders ameliorated by increasingbody mass of the invention can be included in an embodiment eitherindividually or in any combination.

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, practice the present invention toits fullest extent. The foregoing detailed description is given forclearness of understanding only, and no unnecessary limitation should beunderstood therefrom, as modifications within the scope of the inventionmay become apparent to those skilled in the art.

Throughout this application, various publications, patents and publishedpatent applications are cited. The disclosures of these publications,patents and published patent applications referenced in this applicationare herein incorporated by reference in their entirety into the presentdisclosure. Citation herein by Applicant of a publication, patent, orpublished patent application is not an admission by Applicant of saidpublication, patent, or published patent application as prior art.

This application claims the benefit of priority from the followingprovisional patent application, filed via U.S. Express mail with theUnited States Patent and Trademark Office on the indicated date: U.S.Provisional Patent Application No. 60/735,346, filed Nov. 10, 2005. Thedisclosure of the foregoing provisional patent application is hereinincorporated by reference in its entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. By way of illustration and not limitation, FIG. 1 depictsresults from a primary screen of candidate compounds against a “targetreceptor” which is a Gsα Fusion Protein construct of an endogenous,constitutively active Gs-coupled GPCR unrelated to GPR50. Results for“Compound A” are provided in well A2. Results for “Compound “B” areprovided in well G9. (See, Example 6.)

FIG. 2. GPR50 exhibits detectable constitutive activity for lowering alevel of intracellular cAMP. (See, Example 11.)

FIGS. 3A and 3B. Establishment of GPR50-knockout (“deficient”) mice. A.Gene-targeting strategy for generating GPR50-knockout mice. B.Genotyping of GPR50-knockout mice (top panel) and wild-type mice (lowerpanel) by genomic PCR. (See, Example 12.)

FIGS. 4A and 4B. Body weight of GPR50-knockout mice on high fat diet orchow compared to wild-type mice on high fat diet. (See, Example 13.)

FIGS. 5A and 5B. Comparison of body weight of GPR50-knockout mice andwild-type mice on high fat diet. (See, Example 13.)

FIGS. 6A and 6B. Analysis of co-expression of GPR50 by NPY neurons inthe central part of the dorsomedial nucleus of the hypothalamus (DMHc)in rat. A. Representative photomicrographic image illustrating theexpression of GPR50 and NPY in rat DMHc. B. Percentage of NPY neurons inrat DMHc co-expressing GPR50. (See, Example 17.)

FIGS. 7A-7C. Effect of food restriction on GPR50 expression in thecentral part of the dorsomedial nucleus of the hypothalamus (DMHc) inSprague Dawley rats. A. Upper panel. Food intake in ad libitum fed andin food-restricted rats. Lower panel. Percentage original body weight inad libitum fed and in food-restricted rats. B. Representativephotomicrographic images illustrating expression of GPR50 in DMHc in adlibitum fed and in food-restricted rats. C. Relative levels of NPY mRNA(upper panel) and GPR50 mRNA (lower panel) in ad libitum fed and infood-restricted rats. (See, Example 20.)

DETAILED DESCRIPTION Definitions

ADIPOSITY as used herein shall refer to body fat.

AGONIST shall mean an agent (e.g., ligand, candidate compound) that byvirtue of binding to a GPCR activates the GPCR so as to elicit anintracellular response mediated by the GPCR.

AMINO ACID ABBREVIATIONS used herein are set out in Table B:

TABLE B ALANINE ALA A ARGININE ARG R ASPARAGINE ASN N ASPARTIC ACID ASPD CYSTEINE CYS C GLUTAMIC ACID GLU E GLUTAMINE GLN Q GLYCINE GLY GHISTIDINE HIS H ISOLEUCINE ILE I LEUCINE LEU L LYSINE LYS K METHIONINEMET M PHENYLALANINE PHE F PROLINE PRO P SERINE SER S THREONINE THR TTRYPTOPHAN TRP W TYROSINE TYR Y VALINE VAL V

ANTAGONIST shall mean an agent (e.g., ligand, candidate compound) thatbinds, and preferably binds competitively, to a GPCR at about the samesite as an agonist or partial agonist but which does not activate anintracellular response initiated by the active form of the GPCR, and canthereby inhibit the intracellular response by agonist or partialagonist. An antagonist typically does not diminish the baselineintracellular response in the absence of an agonist or partial agonist.

ANTIBODY is intended herein to encompass monoclonal antibody andpolyclonal antibody. Antibodies of the present invention may be preparedby any suitable method known in the art.

BIOLOGICALLY ACTIVE FRAGMENT of a GPCR polypeptide or amino acidsequence shall mean a fragment of the polypeptide or amino acid sequencehaving structural and biochemical functions of a naturally occurringGPCR. In certain embodiments, the biologically active fragment couplesto a G protein. In certain embodiments, the biologically active fragmentbinds to a ligand.

CANDIDATE COMPOUND shall mean a molecule (for example, and notlimitation, a chemical compound) that is amenable to a screeningtechnique and is used interchangeably herein with TEST COMPOUND.

CODON shall mean a grouping of three nucleotides (or equivalents tonucleotides) which generally comprise a nucleoside [adenosine (A),guanosine (G), cytidine (C), uridine (U) and thyridine (T)] coupled to aphosphate group and which, when translated, encodes an amino acid.

COMPOSITION means a material comprising at least one component.

COMPOUND EFFICACY or EFFICACY shall mean the ability of a compound toinhibit or stimulate one or more GPCR functions, e.g. by measurement ofcAMP level in the presence or absence of a candidate compound. Exemplarymeans of measuring compound efficacy are disclosed in the Examplessection of this patent document.

CONDITION RELATED TO OBESITY is intended to include but not be limitedto hypertension, congestive cardiomyopathy, varicosities, pulmonaryembolism, coronary heart disease, stroke, idiopathic intracranialhypertension, meralgia parethetica, dyspnea, obstructive sleep apnea,hypoventilation syndrome, Pickwickian syndrome, asthma, immobility,degenerative osteoarthritis, low back pain, striae distensae or “stretchmarks,” venous stasis of the lower extremities, lymphedema, cellulitis,intertrigo, carbuncles, acanthosis nigricans, skin tags,gastro-esophageal reflux disorder, nonalcoholic fattyliver/steatohepatitis, cholelithiasis, hernias, colon cancer, stressincontinence, obesity-related glomerulopathy, breast and uterine cancer,depression and low self-esteem, impaired quality of life, metabolicsyndrome, insulin resistance, Type 2 diabetes, dyslipidemia,atherosclerosis, hyperandrogenemia in women, polycystic ovariansyndrome, dysmenorrhea, infertility, pregnancy complications, and malehypogonadism. In some embodiments, the condition related to obesity isselected from the group consisting of hypertension, insulin resistance,metabolic syndrome, Type 2 diabetes, dyslipidemia, atherosclerosis,coronary heart disease, and stroke.

CONSTITUTIVELY ACTIVE RECEPTOR shall mean a receptor stabilized in anactive state by means other than through binding of the receptor to itsligand or a chemical equivalent thereof. A constitutively activereceptor may be endogenous or non-endogenous.

CONSTITUTIVELY ACTIVATED RECEPTOR shall mean an endogenous receptor thathas been modified so as to be constitutively active or to be moreconstitutively active.

CONSTITUTIVE RECEPTOR ACTIVATION shall mean activation of a receptor inthe absence of binding to its ligand or a chemical equivalent thereof.

CONTACT or CONTACTING shall mean bringing at least two moietiestogether, whether in an in vitro system or an in vivo system.

DIRECTLY IDENTIFYING or DIRECTLY IDENTIFIED, in relationship to thephrase “candidate compound” or “test compound”, shall mean the screeningof a compound against a G protein-coupled receptor in the absence of aknown ligand (e.g., a known agonist) to the G protein-coupled receptor.

DYSLIPIDEMIA as used herein refers to abnormal concentrations of serumlipids such as HDL (low), LDL (high), VLDL (high), triglycerides (high),lipoprotein (a) (high), free fatty acids (high) and other serum lipids,or combinations thereof.

ENDOGENOUS shall mean a material that a mammal naturally produces.Endogenous in reference to, for example and not limitation, the term“receptor,” shall mean that which is naturally produced by a mammal (forexample, and not limitation, a human). Endogenous shall be understood toencompass allelic variants of a gene as well as the allelic polypeptidevariants so encoded. As used herein, “endogenous GPCR” and “native GPCR”are used interchangeably. By contrast, the term NON-ENDOGENOUS in thiscontext shall mean that which is not naturally produced by a mammal (forexample, and not limitation, a human).

EXPRESSION VECTOR shall mean a DNA sequence that is required for thetranscription of cloned DNA and translation of the transcribed mRNA inan appropriate host cell recombinant for the expression vector. Anappropriately constructed expression vector should contain an origin ofreplication for autonomous replication in host cells, selectablemarkers, a limited number of useful restriction enzyme sites, apotential for high copy number, and active promoters. The cloned DNA tobe transcribed is operably linked to a constitutively or conditionallyactive promoter within the expression vector.

G PROTEIN-COUPLED RECEPTOR FUSION PROTEIN and GPCR FUSION PROTEIN, inthe context of the invention disclosed herein, each mean anon-endogenous protein comprising an endogenous, constitutively activeGPCR or a non-endogenous, constitutively activated GPCR fused to atleast one G protein, most preferably the alpha (α) subunit of such Gprotein (this being the subunit that binds GTP), with the G proteinpreferably being of the same type as the G protein that naturallycouples with endogenous GPCR. In the preferred form, the G protein canbe fused directly to the C-terminus of the GPCR or there may be spacersbetween the two.

HOST CELL shall mean a cell capable of having a vector incorporatedtherein. In the present context, the vector will typically containnucleic acid encoding a GPCR or GPCR fusion protein in operableconnection with a suitable promoter sequence to permit expression of theGPCR or GPCR fusion protein to occur.

IN NEED OF PREVENTION OR TREATMENT as used herein refers to a judgementmade by a caregiver (e.g. physician, nurse, nurse practitioner, etc. inthe case of humans; veterinarian in the case of animals, includingnon-human mammals) that a subject or animal requires or will benefitfrom treatment. This judgement is made based on a variety of factorsthat are in the realm of a caregiver's expertise, but that include theknowledge that the subject or animal is ill, or will be ill, as theresult of a condition that is treatable by the compounds of theinvention.

INHIBIT or INHIBITING, in relationship to the term “response” shall meanthat a response is decreased or prevented in the presence of a compoundas opposed to in the absence of the compound.

INVERSE AGONIST shall mean an agent (e.g., ligand, candidate compound)which binds to a GPCR and which inhibits the baseline intracellularresponse initiated by the active form of the receptor below the normalbase level activity which is observed in the absence of an agonist orpartial agonist.

LIGAND as used herein shall mean a molecule that specifically binds to aGPCR. An endogenous ligand is an endogenous molecule that binds to anative GPCR. A ligand of a GPCR may be, but is not limited to, anagonist, a partial agonist, an inverse agonist or an antagonist of theGPCR.

METABOLIC SYNDROME as defined herein, and according to the AdultTreatment Panel III (ATP III; National Institutes of Health: ThirdReport of the National Cholesterol Education Program Expert Panel onDetection, Evaluation, and Treatment of High Blood Cholesterol in Adults(Adult Treatment Panel III), Executive Summary; Bethesda, Md., NationalInstitutes of Health, National Heart, Lung and Blood Institute, 2001(NIH pub. No 01-3670), occurs when a person meets three or more of fivecriteria related to obesity, hypertriglyceridemia, low HDL cholesterol,high blood pressure, and high fasting glucose.

As used herein, the terms MODULATE or MODIFY are meant to refer to anincrease or decrease in the amount, quality, or effect of a particularactivity, function or molecule.

MODULATOR shall be understood to encompass agonist, partial agonist,inverse agonist and antagonist as hereinbefore defined.

OBESITY, as used herein, is defined as a body-mass index (BMI) of 30.0or greater, in accordance with the WHO classifications of weight[Kopelman, Nature (2000) 404:635-643; the disclosure of which is hereinincorporated by reference in its entirety]. In certain embodiments,obesity is defined on the basis of body fat content: greater than 25% inmales and greater than 30% in females.

OVERWEIGHT, as used herein, is defined as a body mass index (BMI) of27-29.9.

PARTIAL AGONIST shall mean an agent (e.g., ligand, candidate compound)that by virtue of binding to a GPCR activates the GPCR so as to elicitan intracellular response mediated by the GPCR, albeit to a lesserextent or degree than does a full agonist.

PHARMACEUTICAL COMPOSITION shall mean a composition comprising at leastone active ingredient, whereby the composition is amenable toinvestigation for a specified, efficacious outcome in a mammal (forexample, and not limited to a human). Those of ordinary skill in the artwill understand and appreciate the techniques appropriate fordetermining whether an active ingredient has a desired efficaciousoutcome e.g., based upon the needs of the artisan.

POLYNUCLEOTIDE shall refer to RNA, DNA, or RNA/DNA hybrid sequence ofmore than one nucleotide in either single chain or duplex form. Thepolynucleotides of the invention may be prepared by any known method,including synthetic, recombinant, ex vivo generation, or a combinationthereof, as well as utilizing any purification methods known in the art.

POLYPEPTIDE shall refer to a polymer of amino acids without regard tothe length of the polymer. Thus, PEPTIDES, oligopeptides, and proteinsare included within the definition of polypeptide. This term also doesnot specify or exclude post-expression modifications of polypeptides.For example, polypeptides that include the covalent attachment ofglycosyl groups, acetyl groups, phosphate groups, lipid groups and thelike are expressly encompassed by the term polypeptide.

PRIMER is used herein to denote a specific oligonucleotide sequencewhich is complementary to a target nucleotide sequence and used tohybridize to the target nucleotide sequence. A primer serves as aninitiation point for nucleotide polymerization catalyzed by DNApolymerase, RNA polymerase, or reverse transcriptase.

RECEPTOR FUNCTIONALITY shall refer to the normal operation of a receptorto receive a stimulus and moderate an effect in the cell, including, butnot limited to regulating gene transcription, regulating the influx orefflux of ions, effecting a catalytic reaction, and/or modulatingactivity through G-proteins, such as eliciting a second messengerresponse.

SECOND MESSENGER shall mean an intracellular response produced as aresult of receptor activation. A second messenger can include, forexample, inositol 1,4,5-triphosphate (IP₃), diacylglycerol (DAG), cyclicAMP (cAMP), cyclic GMP (cGMP), MAP kinase activity, MAPK/ERK kinasekinase-1 (MEKK1) activity, and Ca²⁺. Second messenger response can bemeasured for a determination of receptor activation. In addition, secondmessenger response can be measured for the identification of candidatecompounds as, for example, inverse agonists, partial agonists, agonists,and antagonists of the receptor.

SELECTIVE GPR50 MODULATOR, as used herein, refers to a modulator ofGPR50 having selectivity for GPR50 receptor over one or more closelyrelated receptors, such as melatonin receptor 1A (MTNR1A) or melatoninreceptor 1B (MTNR1B).

SMALL MOLECULE shall be taken to mean a compound having a molecularweight of less than about 10,000 grams per mole, including a peptide,peptidomimetic, amino acid, amino acid analogue, polynucleotide,polynucleotide analogue, nucleotide, nucleotide analogue, organiccompound or inorganic compound (i.e. including a heterorganic compoundor organometallic compound), and salts, esters and otherpharmaceutically acceptable forms thereof In certain preferredembodiments, small molecules are organic or inorganic compounds having amolecular weight of less than about 5,000 grains per mole. In certainpreferred embodiments, small molecules are organic or inorganiccompounds having molecular weight of less than about 1,000 grams permole. In certain preferred embodiments, small molecules are organic orinorganic compounds having a molecular weight of less than about 500grams per mole.

STIMULATE or STIMULATING, in relationship to the term “response” shallmean that a response is increased in the presence of a compound asopposed to in the absence of the compound.

SUBJECT as used herein shall preferably refer to a mammal, including butnot limited to a mouse, a rat, a rabbit, a pig, a dog, a cat, anon-human primate, a non-human mammal and a human, more preferably to amouse or rat, most preferably to a human.

THERAPEUTICALLY EFFECTIVE AMOUNT as used herein refers to the amount ofactive compound or pharmaceutical agent that elicits the biological ormedicinal response in a tissue, system, animal, subject or human that isbeing sought by a researcher, veterinarian, medical doctor or otherclinician, which includes one or more of the following:

-   -   (1) Preventing the disease; for example, preventing a disease,        condition or disorder in a subject that may be predisposed to        the disease, condition or disorder but does not yet experience        or display the pathology or symptomatology of the disease,    -   (2) Inhibiting the disease; for example, inhibiting a disease,        condition or disorder in a subject that is experiencing or        displaying the pathology or symptomatology of the disease,        condition or disorder (i.e., arresting further development of        the pathology and/or symptomatology), and    -   (3) Ameliorating the disease; for example, ameliorating a        disease, condition or disorder in a subject that is experiencing        or displaying the pathology or symptomatology of the disease,        condition or disorder (i.e., reversing the pathology and/or        symptomatology).

VARIANT as the term is used herein, is a polynucleotide or polypeptidethat differs from a reference polynucleotide or polypeptiderespectively, but retains essential properties. A typical variant of apolynucleotide differs in nucleotide sequence from another, referencepolynucleotide. Changes in the nucleotide sequence of the variant may ormay not alter the amino acid sequence of a polypeptide encoded by thereference polynucleotide. A typical variant of a polypeptide differs inamino acid sequence from another, reference polypeptide. A variant andreference polypeptide may differ in amino acid sequence by one or moresubstitutions, additions, deletions in any combination. A variant of apolynucleotide or polypeptide may be a naturally occurring one such asan ALLELIC VARIANT, or it may be a variant that is not known to occurnaturally. Non-naturally occurring variants of polynucleotides andpolypeptides may be made by mutagenesis techniques or by directsynthesis.

A. Introduction

The order of the following sections is set forth for presentationalefficiency and is not intended, nor should be construed, as a limitationon the disclosure or the claims to follow.

B. Receptor Expression

1. GPCR Polypeptides of Interest

A GPCR of the invention may comprise an amino acid sequence selectedfrom the group consisting of:

-   -   (a) the amino acid sequence of SEQ ID NO: 2;    -   (b) amino acids 2-617 of SEQ ID NO: 2;    -   (c) amino acids 2-617 of SEQ ID NO: 2, wherein the GPCR does not        comprise amino acids 1-617 of SEQ ID NO: 2;    -   (d) the amino acid sequence of (a), (b) or (c), wherein SEQ ID        NO: 2 comprises any combination of a substitution of serine at        amino acid position 493 of SEQ ID NO: 2 with asparagine, a        substitution of threonine at amino acid position 532 of SEQ ID        NO: 2 with alanine, and a substitution of valine at amino acid        position 606 of SEQ ID NO: 2 with isoleucine;    -   (e) the amino acid sequence of SEQ ID NO: 4;    -   (f) amino acids 2-613 of SEQ ID NO: 4;    -   (g) amino acids 2-613 of SEQ ID NO: 4, wherein the GPCR does not        comprise amino acids 1-613 of SEQ ID NO: 4;    -   (h) the amino acid sequence of (i), (ii) or (iii), wherein SEQ        ID NO: 4 comprises any combination of a substitution of        asparagine at amino acid position 493 of SEQ ID NO: 4 with        serine, a substitution of alanine at amino acid position 528 of        SEQ ID NO: 4 with threonine, and a substitution of valine at        amino acid position 602 of SEQ ID NO: 4 with isoleucine;    -   (i) the amino acid sequence of a G protein-coupled receptor        encoded by a polynucleotide that is amplifiable by polymerase        chain reaction (PCR) on a human DNA sample using specific        primers SEQ ID NO: 9 and SEQ ID NO: 10;    -   (j) the amino acid sequence of a G protein-coupled receptor        encoded by a polynucleotide hybridizing at high stringency to        the complement of SEQ ID NO: 1 or SEQ ID NO: 3;    -   (k) the amino acid sequence of a G protein-coupled receptor        having at least about 75%, at least about 80%, at least about        85%, at least about 90% or at least about 95% identity to SEQ ID        NO: 2 or SEQ ID NO: 4;    -   (l) the amino acid sequence of SEQ ID NO: 6;    -   (m) amino acids 2-591 of SEQ ID NO: 6;    -   (n) amino acids 2-591 of SEQ ID NO: 6 wherein the GPCR does not        comprise amino acids 1-591 of SEQ ID NO: 6;    -   (o) the amino acid sequence of a G protein-coupled receptor        encoded by a polynucleotide hybridizing at high stringency to        the complement of SEQ ID NO: 5;    -   (p) the amino acid sequence of a G protein-coupled receptor        having at least about 75%, at least about 80%, at least about        85%, at least about 90% or at least about 95% identity to SEQ ID        NO: 6;    -   (q) the amino acid sequence of SEQ ID NO: 8;    -   (r) amino acids 2-594 of SEQ ID NO: 8;    -   (s) amino acids 2-594 of SEQ ID NO: 8, wherein the GPCR does not        comprise amino acids 1-594 of SEQ ID NO: 8;    -   (t) the amino acid sequence of a G protein-coupled receptor        encoded by a polynucleotide hybridizing at high stringency to        the complement of SEQ ID NO: 7;    -   (u) the amino acid sequence of a G protein-coupled receptor        having at least about 75%, at least about 80%, at least about        85%, at least about 90% or at least about 95% identity to SEQ ID        NO: 8; and    -   (v) the amino acid sequence of a G protein-coupled receptor that        is a constitutively active version of a receptor having SEQ ID        NO: 2 or SEQ ID NO: 4;    -   or a variant or biologically active fragment thereof.

In some embodiments, the GPCR comprises the amino acid sequence of a Gprotein-coupled receptor having at least about 75%, at least about 80%,at least about 85%, at least about 90% or at least about 95% identity toSEQ ID NO: 2 or SEQ ID NO: 4.

In some embodiments, the G protein-coupled receptor having at leastabout 75%, at least about 80%, at least about 85%, at least about 90% orat least about 95% identity to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6or SEQ ID NO: 8 is an endogenous GPCR. In some embodiments, the Gprotein-coupled receptor having at least about 75%, at least about 80%,at least about 85%, at least about 90% or at least about 95% identity toSEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8 is a mammalianendogenous GPCR. In some embodiments, the G protein-coupled receptorhaving at least about 75%, at least about 80%, at least about 85%, atleast about 90% or at least about 95% identity to SEQ ID NO: 2, SEQ IDNO: 4, SEQ ID NO: 6 or SEQ ID NO: 8 is a non-endogenous GPCR.

In some embodiments, the G protein-coupled receptor that is aconstitutively active version of a receptor having SEQ ID NO: 2 or SEQID NO: 4 is an endogenous G protein-coupled receptor. In someembodiments, the G protein-coupled receptor that is a constitutivelyactive version of a receptor having SEQ ID NO: 2 or SEQ ID NO: 4 is anon-endogenous G protein-coupled receptor.

In some embodiments, the human DNA is human cDNA derived from a tissueor cell type that expresses GPR50. In some embodiments, the human cDNAis derived from hypothalamus. In some embodiments, the human cDNA isderived from pituitary.

In some embodiments, a GPCR of the invention is recombinant. In someembodiments, the recombinant GPCR is recombinant human GPR50.

In some embodiments, a GPCR of the invention is endogenous.

In some embodiments, a GPCR of the invention is non-endogenous.

In some embodiments, a GPCR of the invention is a mammalian GPR50.

In some embodiments, that is endogenous is a mammalian GPR50.

In some embodiments, a GPCR of the invention is constitutively active.In some embodiments, an endogenous GPCR of the invention isconstitutively active. In some embodiments, a non-endogenous GPCR of theinvention is constitutively active. In some embodiments, a mammalianGPR50 of the invention is constitutively active. In some embodiments,the mammalian GPR50 is human GPR50. In some embodiments, the human GPR50is SEQ ID NO: 2 or an allele thereof. In some embodiments, the humanGPR50 is SEQ ID NO: 4 or an allele thereof.

In some embodiments, a GPCR of the invention exhibits a detectable levelof constitutive activity. In some embodiments, an endogenous GPCR of theinvention exhibits a detectable level of constitutive activity. In someembodiments, a non-endogenous GPCR of the invention exhibits adetectable level of constitutive activity. In some embodiments, amammalian GPR50 of the invention exhibits a detectable level ofconstitutive activity. In some embodiments, the mammalian GPR50 is humanGPR50. In some embodiments, the human GPR50 is SEQ ID NO: 2 or an allelethereof. In some embodiments, the human GPR50 is SEQ ID NO: 4 or anallele thereof.

In some embodiments, a GPCR that may be used in the subject methods is aconstitutively active version of a receptor having SEQ ID NO: 2. In someembodiments, the constitutively active version of a receptor having SEQID NO: 2 is an endogenous G protein-coupled receptor. In someembodiments, the constitutively active version of a receptor having SEQID NO: 2 is an endogenous G protein-coupled receptor having SEQ ID NO:2. In some embodiments, the constitutively active version of a receptorhaving SEQ ID NO: 2 is a non-endogenous G protein-coupled receptor. Insome embodiments, a GPCR that may be used in the subject methods is aconstitutively active version of a receptor having SEQ ID NO: 4. In someembodiments, the constitutively active version of a receptor having SEQID NO: 4 is an endogenous G protein-coupled receptor. In someembodiments, the constitutively active version of a receptor having SEQID NO: 4 is an endogenous G protein-coupled receptor having SEQ ID NO:4. In some embodiments, the constitutively active version of a receptorhaving SEQ ID NO: 4 is a non-endogenous G protein-coupled receptor.

By way of illustration and not limitation, deletion of an N-terminalmethionine residue or an N-terminal signal peptide is envisioned toprovide a biologically active fragment that may be used in the subjectinvention. In some embodiments, a biologically active fragment of theinvention is a fragment that exhibits a detectable level of constitutiveactivity. In some embodiments, the constitutive activity is for loweringa level of intracellular cAMP. In some embodiments, the constitutiveactivity is for causing melanophore cells to undergo pigmentaggregation. In certain embodiments, a biologically active fragment ofthe invention is a fragment that specifically binds an antibody thatrecognizes a mammalian endogenous GPR50 (an antibody that recognizes anendogenous mammalian GPR50 can be obtained commercially from, e.g.,Advanced Targeting Systems, San Diego, Calif.; and CHEMICONInternational, Inc., Temecula, Calif.) or specifically binds a knownligand of a mammalian endogenous GPR50. In certain embodiments, theknown ligand of the mammalian endogenous GPR50 is an endogenous ligandof the mammalian endogenous GPR50.

An allelic variant of human GPR50 of SEQ ID NO: 2 or SEQ ID NO: 4, ofmouse GPR50 of SEQ ID NO: 6, or of rat GPR50 of SEQ ID NO: 8 isenvisioned to be within the scope of the invention. In some embodiments,a GPCR that may be used in the subject methods may comprise an allelicvariant of SEQ ID NO: 2 or SEQ ID NO: 4.

A variant which is a mammalian ortholog of human GPR50 of SEQ ID NO: 2or SEQ ID NO: 4 is envisioned to be within the scope of the invention.By way of illustration and not limitation, additional to mouse GPR50 andrat GPR50, sheep GPR50 (GenBank) Accession No. NP_(—)001009726),chimpanzee GPR50 (GenBank® Accession No. XP_(—)001136005), and rhesusmonkey GPR50 (GenBank® Accession No. XP_(—)001092026) are envisioned tobe within the scope of the invention.

In certain embodiments, a variant GPCR that may be used in the subjectmethods is a GPCR derived from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6or SEQ ID NO: 8 by substitution, deletion or addition of one or severalamino acids in the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4,SEQ ID NO: 6 or SEQ ID NO: 8, respectively.

In certain embodiments, a variant GPCR that may be used in the subjectmethods is a GPCR derived from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6or SEQ ID NO: 8 by no more than 10 conservative amino acid substitutionsand/or no more than 3 non-conservative amino acid substitutions in theamino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQID NO: 8, respectively. In certain embodiments, arginine, lysine andhistidine may conservatively substitute for each other; glutamic acidand aspartic acid may conservatively substitute for each other;glutamine and asparagine may conservatively substitute for each other;leucine, isoleucine and valine may conservatively substitute for eachother; phenylalanine, tryptophan and tyrosine may conservativelysubstitute for each other; and glycine, alanine, serine, threonine andmethionine may conservatively substitute for each other. The amino acidsubstitutions, amino acid deletions, and amino acid additions may be atany position (e.g., the C- or N-terminus, or at internal positions).

A variant of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO:8having at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 96%, at least about97%, at least about 98%, at least about 99%, at least about 99.1%, atleast about 99.2%, at least about 99.3%, at least about 99.4%, at leastabout 99.5%, at least about 99.6%, at least about 99.7%, at least about99.8%, or at least about 99.9% identity to SEQ ID NO: 2, SEQ ID NO: 4,SEQ ID NO: 6 or SEQ ID NO: 8, respectively, is envisioned to be withinthe scope of the invention. In some embodiments, said variant is avariant of SEQ ID NO: 2. In some embodiments, said variant is a variantof SEQ ID NO: 4. In some embodiments, the variant which is a variant ofSEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8 is a GPCR. Insome embodiments, the variant which is a variant of SEQ ID NO: 2, SEQ IDNO: 4, SEQ ID NO: 6 or SEQ ID NO: 8 is an endogenous GPCR. In someembodiments, the variant that is an endogenous GPCR is a mammalian GPCR.In some embodiments, the variant which is a variant of SEQ ID NO: 2, SEQID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8 is an non-endogenous GPCR. Insome embodiments, the variant which is a variant of SEQ ID NO: 2, SEQ IDNO: 4, SEQ ID NO: 6 or SEQ ID NO: 8 exhibits a detectable level ofconstitutive activity. In some embodiments, the constitutive activity isfor lowering a level of intracellular cAMP. In some embodiments, theconstitutive activity is for causing melanophore cells to undergopigment aggregation. In certain embodiments, the variant which is avariant of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8specifically binds an antibody that recognizes an mammalian endogenousGPR50 (an antibody that recognizes an endogenous mammalian GPR50 can beobtained commercially from, e.g., Advanced Targeting Systems, San Diego,Calif.; and CHEMICON International, Inc., Temecula, Calif.) orspecifically binds a known ligand of an mammalian endogenous GPR50. Incertain embodiments, the known ligand of the mammalian endogenous GPR50is an endogenous ligand of the mammalian endogenous GPR50. Percentidentity can be determined conventionally using known computer programs.

In certain embodiments, a variant GPCR that may be used in the subjectmethods has an amino acid sequence having at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,of at least about 96%, at least about 97%, at least about 98%, at leastabout 99%, at least about 99.1%, at least about 99.2%, at least about99.3%, at least about 99.4%, at least about 99.5%, at least about 99.6%,at least about 99.7%, at least about 99.8%, or at least about 99.9%identity to SEQ ID NO: 2 or SEQ ID NO: 4. By a variant GPCR having, forexample, 95% “identity” to SEQ ID NO: 2 is meant that the amino acidsequence of the variant is identical to amino acids 1-617 of SEQ ID NO:2 except that it may include up to five amino acid alterations per each100 amino acids of SEQ ID NO: 2. Thus, to obtain for example an aminoacid sequence having at least 95% identity to the amino acid sequence ofSEQ ID NO: 2, up to 5% (5 of 100) of the amino acid residues in thesequence may be inserted, deleted, or substituted with another aminoacid compared with amino acids 1-617 of SEQ ID NO: 2. By a variant GPCRhaving, for example, 95% “identity” to SEQ ID NO: 4 is meant that theamino acid sequence of the variant is identical to amino acids 1-613 ofSEQ ID NO: 4 except that it may include up to five amino acidalterations per each 100 amino acids of SEQ ID NO: 4. Thus, to obtainfor example an amino acid sequence having at least 95% identity to theamino acid sequence of SEQ ID NO: 4, up to 5% (5 of 100) of the aminoacid residues in the sequence may be inserted, deleted, or substitutedwith another amino acid compared with amino acids 1-613 of SEQ ID NO: 4.These alternations may occur at the amino or carboxy termini or anywherebetween those terminal positions, interspersed either subjectly amongresidues in the sequence or in one or more contiguous groups within thesequence.

In some embodiments, a variant GPCR that may be used in the subjectmethods is a GPCR encoded by a polynucleotide hybridizing at highstringency to the complement of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5or SEQ ID NO: 7. In some embodiments, the polynucleotide hybridizes athigh stringency to the complement of SEQ ID NO: 1 or SEQ ID NO: 3. Insome embodiments, the variant is an endogenous GPCR. In someembodiments, the variant that is an endogenous GPCR is a mammalian GPCR.In some embodiments, the variant exhibits a detectable level ofconstitutive activity. In some embodiments, the constitutive activity isfor lowering a level of intracellular cAMP. In some embodiments, theconstitutive activity is for causing melanophore cells to undergopigment aggregation. In certain embodiments, the G protein-coupledreceptor encoded by a polynucleotide hybridizing at high stringency tothe complement of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO:7 specifically binds an antibody that recognizes an mammalian endogenousGPR50 (an antibody that recognizes an endogenous mammalian GPR50 can beobtained commercially from, e.g., Advanced Targeting Systems, San Diego,Calif.; and CHEMICON International, Inc., Temecula, Calif.) orspecifically binds a known ligand of an mammalian endogenous GPR50. Incertain embodiments, the known ligand of the mammalian endogenous GPR50is an endogenous ligand of the mammalian endogenous GPR50. Hybridizationtechniques are well known to the skilled artisan. In some embodiments,stringent hybridization conditions include overnight incubation at 42°C. in a solution comprising: 50% formamide, 5×SSC (1×SSC=150 nM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt'ssolution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmonsperm DNA; followed by washing the filter in 0.1×SSC at about 50° C., atabout 55° C., at about 60° C. or at about 65° C.

a. Sequence Identity

In certain embodiments, percent identity is evaluated using the BasicLocal Alignment Search Tool (“BLAST”), which is well known in the art[See, e.g., Karlin and Altschul, Proc Natl Acad Sci USA (1990)87:2264-2268; Altschul et al., J Mol Biol (1990) 215:403-410; Altschulet al, Nature Genetics (1993) 3:266-272; and Altschul et al., NucleicAcids Res (1997) 25:3389-3402; the disclosure of each of which is hereinincorporated by reference in its entirety]. The BLAST programs may beused with the default parameters or with modified parameters provided bythe user. Preferably, the parameters are default parameters.

A preferred method for determining the best overall match between aquery sequence (e.g., the amino acid sequence of SEQ ID NO:2) and asequence to be interrogated, also referred to as a global sequencealignment, can be determined using the FASTDB computer program based onthe algorithm of Brutlag et al. [Comp App Biosci (1990) 6:237-245; thedisclosure of which is herein incorporated by reference in itsentirety]. In a sequence alignment the query and interrogated sequencesare both amino acid sequences. The results of said global sequencealignment is in percent identity. Preferred parameters used in a FASTDBamino acid alignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1,Joining Penalty=20, Randomization Group=25, Length=0, Cutoff Score=1,Window Size-sequence length, Gap Penalty=5, Gap Size Penalty=0.05,Window Size=247 or the length of the interrogated amino acid sequence,whichever is shorter.

If the interrogated sequence is shorter than the query sequence due toN- or C-terminal deletions, not because of internal deletions, theresults, in percent identity, must be manually corrected because theFASTDB program does not account for N- and C-terminal truncations of theinterrogated sequence when calculating global percent identity. Forinterrogated sequences truncated at the N- and C-termini, relative tothe query sequence, the percent identity is corrected by calculating thenumber of residues of the query sequence that are N- and C-terminal ofthe interrogated sequence, that are not matched/aligned with acorresponding interrogated sequence residue, as a percent of the totalbases of the query sequence. Whether a residue is matched/aligned isdetermined by results of the FASTDB sequence alignment. This percentageis then subtracted from the percent identity, calculated by the aboveFASTDB program using the specified parameters, to arrive at a finalpercent identity score. This final percent identity score is what isused for the purposes of the present invention. Only residues to the N-and C-termini of the interrogated sequence, which are notmatched/aligned with the query sequence, are considered for the purposesof manually adjusting the percent identity score. That is, only queryamino acid residues outside the farthest N- and C-terminal residues ofthe interrogated sequence.

For example, a 90 amino acid residue interrogated sequence is alignedwith a 100-residue query sequence to determine percent identity. Thedeletion occurs at the N-terminus of the interrogated sequence andtherefore, the FASTDB alignment does not match/align with the firstresidues at the N-terminus. The 10 unpaired residues represent 10% ofthe sequence (number of residues at the N- and C-termini notmatched/total number of residues in the query sequence) so 10% issubtracted from the percent identity score calculated by the FASTDBprogram. If the remaining 90 residues were perfectly matched, the finalpercent identity would be 90%.

In another example, a 90-residue interrogated sequence is compared witha 100-residue query sequence. This time the deletions are internal sothere are no residues at the N- or C-termini of the interrogatedsequence, which are not matched/aligned with the query. In this case,the percent identity calculated by FASTDB is not manually corrected.Once again, only residue positions outside the N- and C-terminal ends ofthe subject sequence, as displayed in the FASTDB alignment, which arenot matched/aligned with the query sequence are manually corrected. Noother corrections are made for the purposes of the present invention.

b. Fusion Proteins

In certain embodiments, a polypeptide of interest is a fusion protein,and may contain, for example, an affinity tag domain or a reporterdomain. Suitable affinity tags include any amino acid sequence that maybe specifically bound to another moiety, usually another polypeptide,most usually an antibody. Suitable affinity tags include epitope tags,for example, the V5 tag, the FLAG tag, the HA tag (from hemagglutinininfluenza virus), the myc tag, and the like, as is known in the art.Suitable affinity tags also include domains for which, bindingsubstrates are known, e.g., HIS, GST and MBP tags, as is known in theart, and domains from other proteins for which specific bindingpartners, e.g., antibodies, particularly monoclonal antibodies, areavailable. Suitable affinity tags also include any protein-proteininteraction domain, such as a IgG Fe region, which may be specificallybound and detected using a suitable binding partner, e.g. the IgG Fcreceptor. It is expressly contemplated that such a fusion protein maycontain a heterologous N-terminal domain (e.g., an epitope tag) fusedin-frame with a GPCR that has had its N-terminal methionine residueeither deleted or substituted with an alternative amino acid.

Suitable reporter domains include any domain that can report thepresence of a polypeptide. While it is recognized that an affinity tagmay be used to report the presence of a polypeptide using, e.g., alabeled antibody that specifically binds to the tag, light emittingreporter domains are more usually used. Suitable light emitting reporterdomains include luciferase (from, e.g., firefly, Vargula, Renillareniformis or Renilla muelleri), or light emitting variants thereof.Other suitable reporter domains include fluorescent proteins, (frome.g., jellyfish, corals and other coelenterates as such those fromAequoria, Renilla, Ptilosarcus, Stylatula species), or light emittingvariants thereof. Light emitting variants of these reporter proteins arevery well known in the art and may be brighter, dimmer, or havedifferent excitation and/or emission spectra, as compared to a nativereporter protein. For example, some variants are altered such that theyno longer appear green, and may appear blue, cyan, yellow, enhancedyellow red (termed BFP, CFP, YFP eYFP and RFP, respectively) or haveother emission spectra, as is known in the art. Other suitable reporterdomains include domains that can report the presence of a polypeptidethrough a biochemical or color change, such as β-galactosidase,β-glucuronidase, chloramphenicol acetyl transferase, and secretedembryonic alkaline phosphatase.

Also as is known in the art, an affinity tags or a reporter domain maybe present at any position in a polypeptide of interest. However, inmost embodiments, they are present at the C- or N-terminal end of apolypeptide of interest.

2. Nucleic Acids Encoding GPCR Polypeptides of Interest

Since the genetic code and recombinant techniques for manipulatingnucleic acid are known, and the amino acid sequences of GPCRpolypeptides of interest described as above, the design and productionof nucleic acids encoding a GPCR polypeptide of interest is well withinthe skill of an artisan. In certain embodiments, standard recombinantDNA technology (Ausubel, et al, Short Protocols in Molecular Biology,3rd ed., Wiley & Sons, 1995; Sambrook, et al., Molecular Cloning: ALaboratory Manual, Second Edition, (1989) Cold Spring Harbor, N.Y.)methods are used. For example, GPCR coding sequences may be isolatedfrom a library of GPCR coding sequence using any one or a combination ofa variety of recombinant methods that do not need to be describedherein. Subsequent substitution, deletion, and/or addition ofnucleotides in the nucleic acid sequence encoding a protein may also bedone using standard recombinant DNA techniques.

For example, site directed mutagenesis and subcloning may be used tointroduce/delete/substitute nucleic acid residues in a polynucleotideencoding a polypeptide of interest. In other embodiments, PCR may beused. Nucleic acids encoding a polypeptide of interest may also be madeby chemical synthesis entirely from oligonucleotides (e.g., Cello etal., Science (2002) 297:1016-8).

In some embodiments, the codons of the nucleic acids encodingpolypeptides of interest are optimized for expression in cells of aparticular species, particularly a mammalian, e.g., mouse, rat, hamster,non-human primate, or human, species. In some embodiments, the codons ofthe nucleic acids encoding polypeptides of interest are optimized forexpression in cells of a particular species, particularly an amphibianspecies.

a. Vectors

The invention further provides vectors (also referred to as“constructs”) comprising a subject nucleic acid. In many embodiments ofthe invention, the subject nucleic acid sequences will be expressed in ahost after the sequences have been operably linked to an expressioncontrol sequence, including, e.g. a promoter. The subject nucleic acidsare also typically placed in an expression vector that can replicate ina host cell either as an episome or as an integral part of the hostchromosomal DNA. Commonly, expression vectors will contain selectionmarkers, e.g., tetracycline or neomycin, to permit detection of thosecells transformed with the desired DNA sequences (see, e.g., U.S. Pat.No. 4,704,362, which is incorporated herein by reference). Vectors,including single and dual expression cassette vectors are well known inthe art (Ausubel, et al, Short Protocols in Molecular Biology, 3rd ed.,Wiley & Sons, 1995; Sambrook, et al., Molecular Cloning: A LaboratoryManual, Second Edition, (1989) Cold Spring Harbor, N.Y.). Suitablevectors include viral vectors, plasmids, cosmids, artificial chromosomes(human artificial chromosomes, bacterial artificial chromosomes, yeastartificial chromosomes, etc.), mini-chromosomes, and the like.Retroviral, adenoviral and adeno-associated viral vectors may be used.

A variety of expression vectors are available to those in the art forpurposes of producing a polypeptide of interest in a cell and includeexpression vectors which are commercially available (e.g., fromInvitrogen, Carlsbad, Calif.; Clontech, Mountain View, Calif.;Stratagene, La Jolla, Calif.). Commercially available expression vectorsinclude, by way of non-limiting example, CMV promoter-based vectors. Onesuitable expression vector is pCMV. The expression vector may beadenoviral. An exemplary adenoviral vector may be purchased as AdEasy™from Qbiogene (Carlsbad, Calif.) [He T C et al, Proc Natl Acad Sci USA(1998) 95:2509-2514; and U.S. Pat. No. 5,922,576; the disclosure of eachof which is herein incorporated by reference in its entirety]. Othersuitable expression vectors will be readily apparent to those ofordinary skill in the art.

The subject nucleic acids usually comprise an single open reading frameencoding a subject polypeptide of interest, however, in certainembodiments, since the host cell for expression of the polypeptide ofinterest may be a eukaryotic cell, e.g., a mammalian cell, such as ahuman cell, the open reading frame may be interrupted by introns.Subject nucleic acid are typically part of a transcriptional unit whichmay contain, in addition to the subject nucleic acid 3′ and 5′untranslated regions (UTRs) which may direct RNA stability,translational efficiency, etc. The subject nucleic acid may also be partof an expression cassette which contains, in addition to the subjectnucleic acid a promoter, which directs the transcription and expressionof a polypeptide of interest, and a transcriptional terminator.

Eukaryotic promoters can be any promoter that is functional in aeukaryotic host cell, including viral promoters and promoters derivedfrom eukaryotic genes. Exemplary eukaryotic promoters include, but arenot limited to, the following: the promoter of the mouse metallothioneinI gene sequence (Hamer et al., J. Mol. Appl. Gen. 1:273-288, 1982); theTK promoter of Herpes virus (McKnight, Cell 31:355-365, 1982); the SV40early promoter (Benoist et al., Nature (London) 290:304-310, 1981); theyeast gall gene sequence promoter (Johnston et al., Proc. Natl. Acad.Sci. (USA) 79:6971-6975, 1982); Silver et al., Proc. Natl. Acad. Sci.(USA) 81:5951-59SS, 1984), the CMV promoter, the EF-1 promoter,Ecdysone-responsive promoter(s), tetracycline-responsive promoter, andthe like. Viral promoters may be of particular interest as they aregenerally particularly strong promoters. In certain embodiments, apromoter is used that is a promoter of the target pathogen. Promotersfor use in the present invention are selected such that they arefunctional in the cell type (and/or animal) into which they are beingintroduced. In certain embodiments, the promoter is a CMV promoter.

In certain embodiments, a subject vector may also provide for expressionof a selectable marker. Suitable vectors and selectable markers are wellknown in the art and discussed in Ausubel, et al, (Short Protocols inMolecular Biology, 3rd ed., Wiley & Sons, 1995) and Sambrook, et al,(Molecular Cloning: A Laboratory Manual, Third Edition, (2001) ColdSpring Harbor, N.Y.). A variety of different genes have been employed asselectable markers, and the particular gene employed in the subjectvectors as a selectable marker is chosen primarily as a matter ofconvenience. Known selectable marker genes include: the thymidine kinasegene, the dihydrofolate reductase gene, the xanthine-guaninephosphoribosyl transferase gene, CAD, the adenosine deaminase gene, theasparagine synthetase gene, the antibiotic resistance genes, e.g. tetr,ampr, Cmr or cat, kanr or neor (aminoglycoside phosphotransferasegenes), the hygromycin B phosphotransferase gene, and the like.

As mentioned above, polypeptides of interest may be fusion proteins thatcontain an affinity domain and/or a reporter domain. Methods for makingfusions between a reporter or tag and a GPCR, for example, at the C- orN-terminus of the GPCR, are well within the skill of one of skill in theart (e.g. McLean et al, Mol. Pharma. Mol Pharmacol. 1999 56:1182-91;Ramsay et al., Br. J. Pharmacology, 2001, 315-323) and will not bedescribed any further. It is expressly contemplated that such a fusionprotein may contain a heterologous N-terminal domain (e.g., an epitopetag) fused in-frame with a GPCR that has had its N-terminal methionineresidue either deleted or substituted with an alternative amino acid. Itis appreciated that a polypeptide of interest may first be made from anative polypeptide and then operably linked to a suitable reporter/tagas described above.

The subject nucleic acids may also contain restriction sites, multiplecloning sites, primer binding sites, ligatable ends, recombination sitesetc., usually in order to facilitate the construction of a nucleic acidencoding a polypeptide of interest.

b. Host Cells

The invention further provides host cells comprising a vector comprisinga subject nucleic acid. Suitable host cells include prokaryotic, e.g.,bacterial cells (for example E. coli), as well as eukaryotic cells e.g.an animal cell (for example an insect, mammal, fish, amphibian, bird orreptile cell), a plant cell (for example a maize or Arabidopsis cell),or a fungal cell (for example a S. cerevisiae cell). In certainembodiments, any cell suitable for expression of a polypeptide ofinterest-encoding nucleic acid may be used as a host cell. Usually, ananimal host cell line is used, examples of which are as follows: monkeykidney cells (COS cells), monkey kidney CVI cells transformed by SV40(COS-7, ATCC CRL 165 1); human embryonic kidney cells (HEK-293 [“293”],Graham et al. J. Gen Virol. 36:59 (1977)); HEK-293T [“293T”] cells; babyhamster kidney cells (BHK, ATCC CCL 10); chinese hamster ovary-cells(CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. (USA) 77:4216, (1980);Syrian golden hamster cells MCB3901 (ATCC CRL-9595); mouse sertoli cells(TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CVIATCC CCL 70); african green monkey kidney cells (VERO-76, ATCCCRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); caninekidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCCCRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells RepG2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL 51); TRI cells(Mather et al., Annals N. Y. Acad. Sci 383:44-68 (1982)); NIH/3T3 cells(ATCC CRL-1658); and mouse L cells (ATCC CCL-1).

In certain embodiments, melanophores are used. Melanophores are skincells found in lower vertebrates. Relevant materials and methods will befollowed according to the disclosure of U.S. Pat. No. 5,462,856 and U.S.Pat. No. 6,051,386. These patent disclosures are herein incorporated byreference in their entirety.

Additional cell lines will become apparent to those of ordinary skill inthe art, and a wide variety of cell lines are available from theAmerican Type Culture Collection, 10801 University Boulevard, Manassas,Va. 20110-2209.

C. Screening of Candidate Compounds

1. Generic GPCR Screening Assay Techniques

When a G protein receptor becomes active, it binds to a G protein (e.g.Gq, Gs, Gi, Gz, Go) and stimulates the binding of GTP to the G protein.The G protein then acts as a GTPase and slowly hydrolyzes the GTP toGDP, whereby the receptor, under normal conditions, becomes deactivated.However, activated receptors continue to exchange GDP to GTP. Anon-hydrolyzable analog of GTP, [³⁵S]GTPγS, can be used to monitorenhanced binding to membranes which express activated receptors. It isreported that [³⁵S]GTPγS can be used to monitor G protein coupling tomembranes in the absence and presence of ligand. An example of thismonitoring, among other examples well-known and available to those inthe art, was reported by Traynor and Nahorski in 1995. A preferred useof this assay system is for initial screening of candidate compoundsbecause the system is generically applicable to all G protein-coupledreceptors regardless of the particular G protein that interacts with theintracellular domain of the receptor.

2. Specific GPCR Screening Assay Techniques

Once candidate compounds are identified using the “generic” Gprotein-coupled receptor assay (i.e., an assay to select compounds thatare agonists or inverse agonists), in some embodiments further screeningto confirm that the compounds have interacted at the receptor site ispreferred. For example, a compound identified by the “generic” assay maynot bind to the receptor, but may instead merely “uncouple” the Gprotein from the intracellular domain.

a. Gs, Gz and Gi.

Gs stimulates the enzyme adenylyl cyclase. Gi (and Gz and Go), on theother hand, inhibit adenylyl cyclase. Adenylyl cyclase catalyzes theconversion of ATP to cAMP; thus, activated GPCRs that couple the Gsprotein are associated with increased cellular levels of cAMP. On theother hand, activated GPCRs that couple Gi (or Gz, Go) protein areassociated with decreased cellular levels of cAMP. See, generally,“Indirect Mechanisms of Synaptic Transmission,” Chpt. 8, From Neuron ToBrain (3^(rd) Ed.) Nichols, J. G. et al eds. Sinauer Associates, Inc.(1992). Thus, assays that detect cAMP can be utilized to determine if acandidate compound is, e.g., an inverse agonist to the receptor (i.e.,such a compound would decrease the levels of cAMP). A variety ofapproaches known in the art for measuring cAMP can be utilized; in someembodiments a preferred approach relies upon the use of anti-cAMPantibodies in an ELISA-based format. Another type of assay that can beutilized is a whole cell second messenger reporter system assay.Promoters on genes drive the expression of the proteins that aparticular gene encodes. Cyclic AMP drives gene expression by promotingthe binding of a cAMP-responsive DNA binding protein or transcriptionfactor (CREB) that then binds to the promoter at specific sites calledcAMP response elements and drives the expression of the gene. Reportersystems can be constructed which have a promoter containing multiplecAMP response elements before the reporter gene, e.g., β-galactosidaseor luciferase. Thus, an activated Gs-linked receptor causes theaccumulation of cAMP that then activates the gene and expression of thereporter protein. The reporter protein such as β-galactosidase orluciferase can then be detected using standard biochemical assays (Chenet al. 1995).

b. Go and Gq.

Gq and Go are associated with activation of the enzyme phospholipase C,which in turn hydrolyzes the phospholipid PIP₂, releasing twointracellular messengers: diacylglycerol (DAG) and inositol1,4,5-triphosphate (IP₃). Increased accumulation of IP₃ is associatedwith activation of Gq- and Go-associated receptors. See, generally,“Indirect Mechanisms of Synaptic Transmission,” Chpt. 8, From Neuron ToBrain (3^(rd) Ed.) Nichols, J. G. et al eds. Sinauer Associates, Inc.(1992). Assays that detect IP₃ accumulation can be utilized to determineif a candidate compound is, e.g., an inverse agonist to a Gq- orGo-associated receptor (i.e., such a compound would decrease the levelsof IP₃). Gq-associated receptors can also been examined using an AP1reporter assay in that Gq-dependent phospholipase C causes activation ofgenes containing AP1 elements; thus, activated Gq-associated receptorswill evidence an increase in the expression of such genes, wherebyinverse agonists thereto will evidence a decrease in such expression,and agonists will evidence an increase in such expression. Commerciallyavailable assays for such detection are available.

3. GPCR Fusion Protein

The use of an endogenous, constitutively active GPCR or anon-endogenous, constitutively activated GPCR, for use in screening ofcandidate compounds for the direct identification of inverse agonists oragonists provides an interesting screening challenge in that, bydefinition, the receptor is active even in the absence of an endogenousligand bound thereto. Thus, in order to differentiate between, e.g., thenon-endogenous receptor in the presence of a candidate compound and thenon-endogenous receptor in the absence of that compound, with an aim ofsuch a differentiation to allow for an understanding as to whether suchcompound may be an inverse agonist or agonist or have no affect on sucha receptor, in some embodiments it is preferred that an approach beutilized that can enhance such differentiation. In some embodiments, apreferred approach is the use of a GPCR Fusion Protein.

Generally, once it is determined that a non-endogenous GPCR has beenconstitutively activated using the assay techniques set forth above (aswell as others known to the art-skilled), it is possible to determinethe predominant G protein that couples with the endogenous GPCR.Coupling of the G protein to the GPCR provides a signaling pathway thatcan be assessed. In some embodiments it is preferred that screening takeplace using a mammalian or a melanophore expression system, as such asystem will be expected to have endogenous G protein therein. Thus, bydefinition, in such a system, the non-endogenous, constitutivelyactivated GPCR will continuously signal. In some embodiments it ispreferred that this signal be enhanced such that in the presence of,e.g., an inverse agonist to the receptor, it is more likely that it willbe able to more readily differentiate, particularly in the context ofscreening, between the receptor when it is contacted with the inverseagonist.

The GPCR Fusion Protein is intended to enhance the efficacy of G proteincoupling with the GPCR. The GPCR Fusion Protein may be preferred forscreening with either an endogenous, constitutively active GPCR or anon-endogenous, constitutively activated GPCR because such an approachincreases the signal that is generated in such screening techniques.This is important in facilitating a significant “signal to noise” ratio;such a significant ratio is preferred for the screening of candidatecompounds as disclosed herein.

The construction of a construct useful for expression of a GPCR FusionProtein is within the purview of those having ordinary skill in the art.Commercially available expression vectors and systems offer a variety ofapproaches that can fit the particular needs of an investigator.Important criteria in the construction of such a GPCR Fusion Proteinconstruct include but are not limited to, that the GPCR sequence and theG protein sequence both be in-frame (preferably, the sequence for theendogenous GPCR is upstream of the G protein sequence), and that the“stop” codon of the GPCR be deleted or replaced such that uponexpression of the GPCR, the G protein can also be expressed. The GPCRcan be linked directly to the G protein, or there can be spacer residuesbetween the two (preferably, no more than about 12, although this numbercan be readily ascertained by one of ordinary skill in the art). Basedupon convenience, it is preferred to use a spacer. In some embodiments,it is preferred that the G protein that couples to the non-endogenousGPCR will have been identified prior to the creation of the GPCR FusionProtein construct. Because there are only a few G proteins that havebeen identified, it is preferred that a construct comprising thesequence of the G protein (i.e., a universal G protein construct, seeExample 4(a) below) be available for insertion of a GPCR sequencetherein; this provides for further efficiency in the context oflarge-scale screening of a variety of different GPCRs having differentsequences.

As noted above, activated GPCRs that couple to Gi, Gz and Go areexpected to inhibit the formation of cAMP making assays based upon thesetypes of GPCRs challenging [i.e., the cAMP signal decreases uponactivation, thus making the direct identification of, e.g., agonists(which would further decrease this signal) challenging]. As will bedisclosed herein, it has been ascertained that for these types ofreceptors, it is possible to create a GPCR Fusion Protein that is notbased upon the GPCR's endogenous G protein, in an effort to establish aviable cyclase-based assay. Thus, for example, an endogenous Gi coupledreceptor can be fused to a Gs protein—such a fusion construct, uponexpression, “drives” or “forces” the endogenous GPCR to couple with,e.g., Gs rather than the “natural” Gi protein, such that a cyclase-basedassay can be established. Thus, for Gi, Gz and Go coupled receptors, insome embodiments it is preferred that when a GPCR Fusion Protein is usedand the assay is based upon detection of adenylyl cyclase activity, thatthe fusion construct be established with Gs (or an equivalent G proteinthat stimulates the formation of the enzyme adenylyl cyclase).

TABLE C Effect of cAMP Production Effect of IP₃ Accumulation Effect ofcAMP Effect on IP₃ upon Activation of GPCR upon Activation of GPCRProduction upon Accumulation upon G (i.e., constitutive activa- (i.e.,constitutive activa- contact with an contact with an protein tion oragonist binding) tion or agonist binding) Inverse Agonist InverseAgonist Gs Increase N/A Decrease N/A Gi Decrease N/A Increase N/A GzDecrease N/A Increase N/A Go Decrease Increase Increase Decrease Gq N/AIncrease N/A Decrease

Equally effective is a G Protein Fusion construct that utilizes a GqProtein fused with a Gs, Gi, Gz or Go Protein. In some embodiments apreferred fusion construct can be accomplished with a Gq Protein whereinthe first six (6) amino acids of the G-protein α-subunit (“Gαq”) isdeleted and the last five (5) amino acids at the C-terminal end of Gαqis replaced with the corresponding amino acids of the Gα of the Gprotein of interest. For example, a fusion construct can have a Gq (6amino acid deletion) fused with a Gi Protein, resulting in a “Gq/GiFusion Construct”. This fusion construct will force the endogenous Gicoupled receptor to couple to its non-endogenous G protein, Gq, suchthat the second messenger, for example, inositol triphosphate ordiacylglycerol, can be measured in lieu of cAMP production.

4. Co-Transfection of a Target Gi Coupled GPCR with a Signal-Enhancer GsCoupled GPCR (cAMP Based Assays)

A Gi coupled receptor is known to inhibit adenylyl cyclase, and,therefore, decreases the level of cAMP production, which can make theassessment of cAMP levels challenging. In certain embodiments, aneffective technique in measuring the decrease in production of cAMP asan indication of activation of a receptor that predominantly couples Giupon activation can be accomplished by co-transfecting a signalenhancer, e.g., a non-endogenous, constitutively activated receptor thatpredominantly couples with Gs upon activation (e.g., TSHR-A623I; seeinfra), with the Gi linked GPCR. As is apparent, activation of a Gscoupled receptor can be determined based upon an increase in productionof cAMP. Activation of a Gi coupled receptor leads to a decrease inproduction cAMP. Thus, the co-transfection approach is intended toadvantageously exploit these “opposite” affects. For example,co-transfection of a non-endogenous, constitutively activated Gs coupledreceptor (the “signal enhancer”) with expression vector alone provides abaseline cAMP signal (i.e., although the Gi coupled receptor willdecrease cAMP levels, this “decrease” will be relative to thesubstantial increase in cAMP levels established by constitutivelyactivated Gs coupled signal enhancer). By then co-transfecting thesignal enhancer with the “target receptor”, an inverse agonist of the Gicoupled target receptor will increase the measured cAMP signal, while anagonist of the Gi coupled target receptor will decrease this signal.

Candidate compounds that are directly identified using this approachshould be assessed independently to ensure that these do not target thesignal enhancing receptor (this can be done prior to or after screeningagainst the co-transfected receptors).

D. Medicinal Chemistry

Candidate Compounds

Any molecule known in the art can be tested for its ability to modulate(increase or decrease) the activity of a GPCR of the present invention.For identifying a compound that modulates activity, candidate compoundscan be directly provided to a cell expressing the receptor.

This embodiment of the invention is well suited to screen chemicallibraries for molecules which modulate, e.g., inhibit, antagonize, oragonize, the amount of, or activity of, a receptor. The chemicallibraries can be peptide libraries, peptidomimetic libraries, chemicallysynthesized libraries, recombinant, e.g., phage display libraries, andin vitro translation-based libraries, other non-peptide syntheticorganic libraries, etc. This embodiment of the invention is also wellsuited to screen endogenous candidate compounds comprising biologicalmaterials, including but not limited to plasma and tissue extracts, andto screen libraries of endogenous compounds known to have biologicalactivity.

In some embodiments, direct identification of candidate compounds isconducted in conjunction with compounds generated via combinatorialchemistry techniques, whereby thousands of compounds are randomlyprepared for such analysis. The candidate compound may be a member of achemical library. This may comprise any convenient number of subjectmembers, for example tens to hundreds to thousand to millions ofsuitable compounds, for example peptides, peptoids and other oligomericcompounds (cyclic or linear), and template-based smaller molecules, forexample benzodiazepines, hydantoins, biaryls, carbocyclic and polycycliccompounds (e.g., naphthalenes, phenothiazines, acridines, steroidsetc.), carbohydrate and amino acid derivatives, dihydropyridines,benzhydryls and heterocycles (e.g., trizines, indoles, thiazolidinesetc.). The numbers quoted and the types of compounds listed areillustrative, but not limiting. Preferred chemical libraries comprisechemical compounds of low molecular weight and potential therapeuticagents.

Exemplary chemical libraries are commercially available from severalsources (ArQule, Tripos/PanLabs, ChemDesign, Pharmacopoeia). In somecases, these chemical libraries are generated using combinatorialstrategies that encode the identity of each member of the library on asubstrate to which the member compound is attached, thus allowing directand immediate identification of a molecule that is an effectivemodulator. Thus, in many combinatorial approaches, the position on aplate of a compound specifies that compound's composition. Also, in oneexample, a single plate position may have from 1-20 chemicals that canbe screened by administration to a well containing the interactions ofinterest. Thus, if modulation is detected, smaller and smaller pools ofinteracting pairs can be assayed for the modulation activity. By suchmethods, many candidate molecules can be screened.

Many diversity libraries suitable for use are known in the art and canbe used to provide compounds to be tested according to the presentinvention. Alternatively, libraries can be constructed using standardmethods. Further, more general, structurally constrained, organicdiversity (e.g., nonpeptide) libraries, can also be used. By way ofexample, a benzodiazepine library (see e.g., Bunin et al., 1994, Proc.Natl. Acad. Sci. USA 91:4708-4712) may be used.

In another embodiment of the present invention, combinatorial chemistrycan be used to identify modulators of the GPCRs of the presentinvention. Combinatorial chemistry is capable of creating librariescontaining hundreds of thousands of compounds, many of which may bestructurally similar. While high throughput screening programs arecapable of screening these vast libraries for affinity for knowntargets, new approaches have been developed that achieve libraries ofsmaller dimension but which provide maximum chemical diversity. (Seee.g., Matter, 1997, Journal of Medicinal Chemistry 40:1219-1229).

One method of combinatorial chemistry, affinity fingerprinting, haspreviously been used to test a discrete library of small molecules forbinding affinities for a defined panel of proteins. The fingerprintsobtained by the screen are used to predict the affinity of the subjectlibrary members for other proteins or receptors of interest (in theinstant invention, the receptors of the present invention). Thefingerprints are compared with fingerprints obtained from othercompounds known to react with the protein of interest to predict whetherthe library compound might similarly react. For example, rather thantesting every ligand in a large library for interaction with a complexor protein component, only those ligands having a fingerprint similar toother compounds known to have that activity could be tested. (See, e.g.,Kauvar et al., 1995, Chemistry and Biology 2:107-118; Kauvar, 1995,Affinity fingerprinting, Pharmaceutical Manufacturing International.8:25-28; and Kauvar, Toxic-Chemical Detection by Pattern Recognition inNew Frontiers in Agrochemical Immunoassay, D. Kurtz. L. Stanker and J.H. Skerritt. Editors, 1995, AOAC: Washington, D.C., 305-312).

In some embodiments, the candidate compound is a polypeptide. In somepreferred embodiments, the candidate compound is a small molecule. Insome embodiments, the candidate compound is not an antibody or anantigen-binding fragment thereof.

Candidate Compounds Identified as Modulators

Generally, the results of such screening will be compounds having uniquecore structures; thereafter, these compounds may be subjected toadditional chemical modification around a preferred core structure(s) tofurther enhance the medicinal properties thereof. Such techniques areknown to those in the art and will not be addressed in detail in thispatent document.

In certain embodiments, a modulator of the invention is orally active. Anumber of computational approaches available to those of ordinary skillin the art have been developed for prediction of oral bioavailability ofa drug [Ooms et al., Biochim Biophys Acta (2002) 1587:118-25; Clark &Grootenhuis, Curr Opin Drug Discov Devel (2002) 5:382-90; Cheng et al.,J Comput Chem (2002) 23:172-83; Norinder & Haeberlein, Adv Drug DelivRev (2002) 54:291-313; Matter et al., Comb Chem High Throughput Screen(2001) 4:453-75; Podlogar & Muegge, Curr Top Med Chem (2001) 1:257-75;the disclosure of each of which is herein incorporated by reference inits entirety). Furthermore, positron emission tomography (PET) has beensuccessfully used by a number of groups to obtain direct measurements ofdrug distribution, including an assessment of oral bioavailability, inthe mammalian body following oral administration of the drug, includingnon-human primate and human body [Noda et al., J Nucl Med (2003)44:105-8; Gulyas et al., Eur J Nucl Med Mol Imaging (2002) 29:1031-8;Kanerva et al., Psychopharmacology (1999) 145:76-81; the disclosure ofeach of which is herein incorporated by reference in its entirety]. Insome embodiments, a modulator of the invention is orally active.

In certain embodiments, a modulator of the invention which is orallyactive is able to cross the blood-brain barrier. A number ofcomputational approaches available to those of ordinary skill in the arthave been developed for prediction of the permeation of the blood-brainbarrier [Ooms et al., Biochim Biophys Acta (2002) 1587:118-25; Clark &Grootenhuis, Curr Opin Drug Discov Devel (2002) 5:382-90; Cheng et al.,J Comput Chem (2002) 23:172-83; Norinder & Haeberlein, Adv Drug DelivRev (2002) 54:291-313; Matter et al., Comb Chem High Throughput Screen(2001) 4:453-75; Podlogar & Muegge, Curr Top Med Chem (2001) 1:257-75;the disclosure of each of which is herein incorporated by reference inits entirety). A number of in vitro methods have been developed topredict blood-brain barrier permeability of drugs [Lohmann et al., JDrug Target (2002) 10:263-76; Hansen et al., J Pharm Biomed Anal (2002)27:945-58; Otis et al., J Pharmocol Toxicol Methods (2001) 45:71-7;Dehouck et al, J Neurochem (1990) 54:1798-801; the disclosure of each ofwhich is herein incorporated by reference in its entirety]. Furthermore,a number of strategies have been developed to enhance drug deliveryacross the blood-brain barrier [Scherrmann, Vascul Pharmacol (2002)38:349-54; Pardridge, Arch Neurol (2002) 59:35-40; Pardridge, Neuron(2002) 36:555-8; the disclosure of each of which is hereby incorporatedby reference in its entirety]. Finally, positron emission tomography(PET) has been successfully used by a number of groups to obtain directmeasurements of drug distribution, including that within brain, in themammalian body, including non-human primate and human body [Noda et al.,J Nucl Med (2003) 44:105-8; Gulyas et al., Eur 3 Nucl Med Mol Imaging(2002) 29:1031-8; Kanerva et al., Psychopharmacology (1999) 145:76-81;the disclosure of each of which is herein incorporated by reference inits entirety].

In some embodiments, said modulator is selective for GPR50, wherein amodulator selective for GPR50 is understood to refer to a modulatorhaving selectivity for GPR50 over one or more closely related receptors,such as melatonin receptor 1A (MTNR1A; GenBank® Accession No.NP_(—)005949) or melatonin receptor 1B (MTNR1B; GenBank® Accession No.NP_(—)005950). In certain embodiments, a GPR50 selective modulator is aGPR50 selective inverse agonist or antagonist having a selectivity forGPR50 over MTNR1A or MTNR1B of at least about 10-fold or of at leastabout 100-fold. In certain embodiments, a GPR50 selective modulator is aGPR50 selective inverse agonist or antagonist having a selectivity forGPR50 over MTNR1A and MTNR1B of at least about 10-fold or of at leastabout 100-fold. In some preferred embodiments, GPR50 is human GPR50.

In some embodiments, the modulator is an inverse agonist or antagonistwith an IC₅₀ of less than about 10 μM, of less than about 1 μM, of lessthan about 100 nM, or of less than about 10 nM at human, mouse or ratGPR50, preferably at human GPR50. In some embodiments, the modulator isan inverse agonist or antagonist with an IC₅₀ of less than a valueselected from the interval of about 10 nM to 10 μM. In some embodiments,modulator is an inverse agonist or antagonist with an IC₅₀ of less thana value selected from the interval of about 10 nM to 1 μM. In someembodiments, the modulator is an inverse agonist or antagonist with anIC₅₀ of less than a value selected from the interval of about 10 nM to100 nM. In some embodiments, the modulator is an inverse agonist orantagonist with an IC₅₀ of less than about 10 μM, of less than about 1μM, of less than about 100 nM, or of less than about 10 nM in GTPγSbinding assay carried out with membrane from transfected CHO cells, orin pigment aggregation assay carried out in transfected melanophores, orin cAMP assay carried out in transfected 293 cells optionallyco-transfected with TSHR, wherein the transfected CHO cells or thetransfected melanophore cells or the transfected 293 cells express arecombinant GPR50 having an amino acid sequence selected from SEQ ID NO:2, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8. In some embodiments, therecombinant GPR50 has the amino acid sequence of SEQ ID NO: 2. In someembodiments, the recombinant GPR50 has the amino acid sequence of SEQ IDNO: 4. In some embodiments, the modulator is an inverse agonist orantagonist with an IC₅₀ of less than about 10 μM, of less than about 1μM, of less than about 100 nM, or of less than about 10 nM in saidassay. In some embodiments, said modulator is an inverse agonist orantagonist with an IC₅₀ of less than 10 μM in said assay, of less than 9μM in said assay, of less than 8 μM in said assay, of less than 7 μM insaid assay, of less than 6 μM in said assay, of less than 5 μM in saidassay, of less than 4 μM in said assay, of less than 3 μM in said assay,of less than 2 μM in said assay, of less than 1 μM in said assay, ofless than 900 nM in said assay, of less than 800 nM in said assay, ofless than 700 nM in said assay, of less than 600 nM in said assay, ofless than 500 nM in said assay, of less than 400 nM in said assay, ofless than 300 nM in said assay, of less than 200 nM in said assay, ofless than 100 nM in said assay, of less than 90 nM in said assay, ofless than 80 nM in said assay, of less than 70 nM in said assay, of lessthan 60 nM in said assay, of less than 50 nM in said assay, of less than40 nM n said assay, of less than 30 nM in said assay, of less than 20 nMin said assay, or of less than 10 nM in said assay. In some embodiments,the modulator is an inverse agonist or antagonist with an IC₅₀ in saidassay of less than a value selected from the interval of about 10 nM to10 μM. In some embodiments, the modulator is an inverse agonist orantagonist with an IC₅₀ in said assay of less than a value selected fromthe interval of about 10 nM to 1 μM. In some embodiments, the modulatoris an inverse agonist or antagonist with an IC₅₀ in said assay of lessthan a value selected from the interval of about 10 nM to 100 nM.

In some embodiments, the modulator is an agonist or partial agonist withan EC₅₀ of less than about 10 μM, of less than about 1 μM, of less thanabout 100 nM, or of less than about 10 nM at human, mouse or rat GPR50,preferably at human GPR50. In some embodiments, the modulator is anagonist or partial agonist with an EC₅₀ of less than a value selectedfrom the interval of about 10 nM to 10 μM. In some embodiments,modulator is an agonist or partial agonist with an EC₅₀ of less than avalue selected from the interval of about 10 nM to 1 μM. In someembodiments, the modulator is an agonist or partial agonist with an EC₅₀of less than a value selected from the interval of about 10 nM to 100nM. In some embodiments, the modulator is an agonist or partial agonistwith an EC₅₀ of less than about 10 μM, of less than about 1 μM, of lessthan about 100 nM, or of less than about 10 nM in GTPγS binding assaycarried out with membrane from transfected CHO cells, or in pigmentaggregation assay carried out in transfected melanophores, or in cAMPassay carried out in transfected 293 cells optionally co-transfectedwith TSHR, wherein the transfected CHO cells or the transfectedmelanophore cells or the transfected 293 cells express a recombinantGPR50 having an amino acid sequence selected from SEQ ID NO: 2, SEQ IDNO: 4, SEQ ID NO: 6 and SEQ ID NO: 8. In some embodiments, therecombinant GPR50 has the amino acid sequence of SEQ ID NO: 2. In someembodiments, the recombinant GPR50 has the amino acid sequence of SEQ IDNO: 4. In some embodiments, the modulator is an agonist or partialagonist with an EC₅₀ of less than about 10 μM, of less than about 1 μM,of less than about 100 nM, or of less than about 10 nM in said assay. Insome embodiments, said modulator is an agonist or partial agonist withan EC₅₀ of less than 10 μM in said assay, of less than 9 μM in saidassay, of less than 8 μM in said assay, of less than 7 μM in said assay,of less than 6 μM in said assay, of less than 5 μM in said assay, ofless than 4 μM in said assay, of less than 3 μM in said assay, of lessthan 2 μM in said assay, of less than 1 μM in said assay, of less than900 nM in said assay, of less than 800 nM in said assay, of less than700 nM in said assay, of less than 600 nM in said assay, of less than500 nM in said assay, of less than 400 nM in said assay, of less than300 nM in said assay, of less than 200 nM in said assay, of less than100 nM in said assay, of less than 90 nM in said assay, of less than 80nM in said assay, of less than 70 nM in said assay, of less than 60 nMin said assay, of less than 50 nM in said assay, of less than 40 nM nsaid assay, of less than 30 nM in said assay, of less than 20 nM in saidassay, or of less than 10 nM in said assay. In some embodiments, themodulator is an agonist or partial agonist with an EC₅₀ in said assay ofless than a value selected from the interval of about 10 nM to 10 μM. Insome embodiments, the modulator is an agonist or partial agonist with anEC₅₀ in said assay of less than a value selected from the interval ofabout 10 nM to 1 μM. In some embodiments, the modulator is an agonist orpartial agonist with an EC₅₀ in said assay of less than a value selectedfrom the interval of about 10 nM to 100 nM.

E. Pharmaceutical Compositions

Compounds of the invention can be formulated into pharmaceuticalcompositions using techniques well known in the art.

The invention provides methods of treatment (and prevention) byadministration to a subject in need of said treatment (or prevention) atherapeutically effect amount of a modulator or a ligand of theinvention [also see, e.g., PCT Application Number PCT/IB02/01461published as WO 02/066505 on 29 Aug. 2002; the disclosure of each ofwhich is herein incorporated by reference in its entirety]. In oneaspect, the modulator or the ligand is a small molecule. In one aspect,the modulator is an an inverse agonist or an antagonist. In one aspect,the modulator is an inverse agonist. In one aspect, the modulator is anantagonist. In one aspect, the modulator is substantially purified. Inone aspect, the subject is a mammal including, but not limited to cows,pigs, horses, non-human primates, cats, dogs, rabbits, rats, mice, etc.,and is preferably a human.

Modulators of the invention can be administered to non-human mammals[see Examples, infra] and/or humans, alone or in pharmaceuticalcompositions where they are mixed with suitable carriers or excipient(s)using techniques well known to those in the art. Suitablepharmaceutically-acceptable carriers are available to those in the art;for example, see Remington's Pharmaceutical Sciences, 16^(th) Edition,1980, Mack Publishing Co., (Oslo et al., eds.).

The pharmaceutical composition is then provided at a therapeuticallyeffective dose. A therapeutically effective dose refers to that amountof a modulator sufficient to result in prevention or amelioration ofsymptoms or physiological status of a disorder as determinedillustratively and not by limitation by the methods described herein,wherein the prevention or amelioration of symptoms or physiologicalstatus of a disorder includes but is not limited to decreasing body massin a subject, decreasing adiposity in a subject, decreasing percentagebody fat in a subject, and preventing or treating obesity or a conditionrelated thereto.

It is expressly considered that the modulators of the invention may beprovided alone or in combination with other pharmaceutically orphysiologically acceptable compounds. Other compounds for the treatmentof disorders of the invention, wherein the treatment of disorders of theinvention includes but is not limited to decreasing body mass in asubject, decreasing percentage body fat in a subject, and preventing ortreating obesity or a condition related thereto.

While the compounds of the invention can be administered as the soleactive pharmaceutical agent (i.e., mono-therapy), compounds of theinvention can also be used in combination with other pharmaceuticalagents (i.e., combination-therapy) for the treatment of thediseases/conditions/disorders described herein. Therefore, anotheraspect of the present invention includes methods of treatment comprisingadministering to a subject in need of treatment a therapeuticallyeffective amount of an antagonist or an inverse agonist of the presentinvention in combination with one or more additional pharmaceuticalagent as described herein.

It will be understood that the scope of combination-therapy of thecompounds of the present invention with other pharmaceutical agents isnot limited to those listed herein, supra or infra, but includes inprinciple any combination with any pharmaceutical agent orpharmaceutical composition useful for the treatment diseases, conditionsor disorders of the present invention in a subject.

In one aspect of the present invention, the other pharmaceutically orphysiologically acceptable compound is an anti-obesity agent such asapolipoprotein-B secretion/microsomal triglyceride transfer protein(apo-B/MTP) inhibitors, MCR4 agonists, cholescystokinin-A (CCK-A)agonists, serotonin and norepinephrine reuptake inhibitors (for example,sibutramine), sympathomimetic agents, β3 adrenergic receptor agonists,dopamine agonists (for example, bromocriptine), melanocyte-stimulatinghormone receptor analogs, 5-HT_(2c) serotonin receptor agonists (forexample, lorcaserin hydrochloride), cannabinoid 1 receptor antagonists[for example, SR141716:N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)4-methyl-1H-pyrazole-3-carboxamide],melanin concentrating hormone antagonists, leptons (the OB protein),leptin analogues, leptin receptor agonists, galanin antagonists, lipaseinhibitors (such as tetrahydrolipstatin, i.e., Orlistat), anorecticagents (such as a bombesin agonist), Neuropeptide-Y antagonists,thromimetic agents, dehydroepiandrosterone or an analogue thereof,glucocorticoid receptor agonists or antagonists, orexin receptorantagonists, urocortin binding protein antagonists, glucagon-likepeptide-1 receptor agonists, ciliary neutrotrophic factors (such asAxokine™ available from Regeneron Pharmaceuticals, Inc., Tarrytown, N.Y.and Procter & Gamble Company, Cincinnati, Ohio), human agouti-relatedprotein (AGRP) antagonists, ghrelin receptor antagonists, histamine 3receptor antagonists or reverse agonists, neuromedin U receptoragonists, noradrenergic anorectic agents (for example, phentermine,mazindol and the like) and appetite suppressants (for example,bupropion). In some embodiments, the anti-obesity agent is selected fromthe group consisting of orlistat, sibutramine, bromocriptine, ephedrine,leptin, and pseudoephedrine.

In accordance to an aspect of the present invention, a compound of thepresent invention can be used in combination with a pharmaceutical agentor agents belonging to one or more of the classes of drugs cited herein.

Routes of Administration

Suitable routes of administration include oral, nasal, rectal,transmucosal, transdermal, or intestinal administration, parenteraldelivery, including intramuscular, subcutaneous, intramedullaryinjections, as well as intrathecal, direct intraventricular,intravenous, intraperitoneal, intranasal, intrapulmonary (inhaled) orintraocular injections using methods known in the art. Otherparticularly preferred routes of administration are aerosol and depotformulation. Sustained release formulations, particularly depot, of theinvented medicaments are expressly contemplated. In certain embodiments,route of administration is oral.

Composition/Formulation

Pharmaceutical or physiologically acceptable compositions andmedicaments for use in accordance with the present invention may beformulated in a conventional manner using one or more physiologicallyacceptable carriers comprising excipients and auxiliaries. Properformulation is dependent upon the route of administration chosen.

Certain of the medicaments described herein will include apharmaceutically or physiologically acceptable carrier and at least onemodulator of the invention. For injection, the agents of the inventionmay be formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks's solution, Ringer's solution, orphysiological saline buffer such as a phosphate or bicarbonate buffer.For transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants aregenerally known in the art.

Pharmaceutical or physiologically acceptable preparations that can betaken orally include push-fit capsules made of gelatin, as well as soft,sealed capsules made of gelatin and a plasticizer, such as glycerol orsorbitol. The push-fit capsules can contain the active ingredients inadmixture with fillers such as lactose, binders such as starches, and/orlubricants such as talc or magnesium stearate and, optionally,stabilizers. In soft capsules, the active compounds may be dissolved orsuspended in suitable liquids, such as fatty oils, liquid paraffin, orliquid polyethylene glycols. In addition, stabilizers may be added. Allformulations for oral administration should be in dosages suitable forsuch administration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs for a nebulizer, with the useof a suitable gaseous propellant, e.g., carbon dioxide. In the case of apressurized aerosol the dosage unit may be determined by providing avalve to deliver a metered amount. Capsules and cartridges of, e.g.,gelatin, for use in an inhaler or insufflator, may be formulatedcontaining a powder nix of the compound and a suitable powder base suchas lactose or starch.

The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage for, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspension, solutions or emulsions in aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Pharmaceutical or physiologically acceptable formulations for parenteraladministration include aqueous solutions of the active compounds inwater-soluble form. Aqueous suspension may contain substances thatincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension may alsocontain suitable stabilizers or agents that increase the solubility ofthe compounds to allow for the preparation of highly concentratedsolutions.

Alternatively, the active ingredient may be in powder or lyophilizedform for constitution with a suitable vehicle, such as sterilepyrogen-free water, before use.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

In a particular embodiment, the compounds can be delivered via acontrolled release system. In one embodiment, a pump may be used(Langer, supra; Sefton, 1987, CRC Crit. Ref Biomed. Eng. 14:201-240;Buchwald et al., 1980, Surgery 88:507-516; Saudek et al., 1989, N. Engl.J. Med. 321:574-579). In another embodiment, polymeric materials can beused (Medical Applications of Controlled Release, Langer and Wise, eds.,CRC Press, Boca Raton, Fla., 1974; Controlled Drug Bioavailability, DrugProduct Design and Performance, Smolen and Ball, eds., Wiley, New York,1984; Ranger and Peppas, 1983, Macromol. Sci. Rev. Macromol. Chem.23:61; Levy et al., 1985, Science 228:190-192; During et al., 1989, Ann.Neurol. 25:351-356; Howard et al., 1989, J. Neurosurg. 71:858-863).Other controlled release systems are discussed in the review by Langer(1990, Science 249:1527-1533).

Additionally, the compounds may be delivered using a sustained-releasesystem, such as semipermeable matrices of solid hydrophobic polymerscontaining the therapeutic agent. Various sustained release materialshave been established and are well known by those skilled in the art.Sustained-release capsules may, depending on their chemical nature,release the compounds for a few weeks up to over 100 days.

Depending on the chemical nature and the biological stability of thetherapeutic reagent, additional strategies for modulator stabilizationmay be employed.

The pharmaceutical or physiologically acceptable compositions also maycomprise suitable solid or gel phase carriers or excipients. Examples ofsuch carriers or escipients include but are not limited to calciumcarbonate, calcium phosphate, various sugars, starches, cellulosderivatives, gelatin, and polymers such as polyethylene glycols.

Effective Dosage

Pharmaceutical or physiologically acceptable compositions suitable foruse in the present invention include compositions wherein the activeingredients are contained in an effective amount to achieve theirintended purpose. More specifically, a therapeutically effective amountmeans an amount effective to prevent development of or to alleviate theexisting symptoms of the subject being treated. Determination of theeffective amounts is well within the capability of those skilled in theart, especially in light of the detailed disclosure provided herein.

For any compound used in the method of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. For example, a dose can be formulated in animal modelsto achieve a circulating concentration range that includes orencompasses a concentration point or range shown to increase anintracellular level of cAMP in a cell comprising GPR50 in an in vitroassay. Such information can be used to more accurately determine usefuldoses in humans.

A therapeutically effective dose refers to that amount of the compoundthat results in amelioration of symptoms in a patient. Toxicity andtherapeutic efficacy of such compounds can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., for determining the LD₅₀ (the dose lethal to 50% of the testpopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe test population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratiobetween LD₅₀ and ED₅₀. Compounds that exhibit high therapeutic indicesare preferred.

The data obtained from these cell culture assays and animal studies canbe used in formulating a range of dosage for use in humans. The dosageof such compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀, with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. The exactformulation, route of administration and dosage can be chosen by thesubject physician in view of the patient's condition. (See, e.g., Finglet al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch. 1).

Dosage amount and interval may be adjusted subjectly to provide plasmalevels of the active compound which are sufficient to prevent or treat adisorder of the invention, depending on the particular situation.Dosages necessary to achieve these effects will depend on subjectcharacteristics and route of administration.

Dosage intervals can also be determined using the value for the minimumeffective concentration. Compounds should be administered using aregimen that maintains plasma levels above the minimum effectiveconcentration for 10-90% of the time, preferably between 30-99%, andmost preferably between 50-90%. In cases of local administration orselective uptake, the effective local concentration of the drug may notbe related to plasma concentration.

The amount of composition administered will, of course, be dependent onthe subject being treated, on the subject's weight, the severity of theaffliction, the manner of administration, and the judgement of theprescribing physician.

A preferred dosage range for the amount of a modulator of the invention,which can be administered on a daily or regular basis to achieve desiredresults is 0.1-100 mg/kg body mass. Other preferred dosage range is0.1-30 mg/kg body mass. Other preferred dosage range is 0.1-10 mg/kgbody mass. Other preferred dosage range is 0.1-3.0 mg/kg body mass. Ofcourse, these daily dosages can be delivered or administered in smallamounts periodically during the course of a day. It is noted that thesedosage ranges are only preferred ranges and are not meant to be limitingto the invention. Said desired results include, but are not limited to,decreasing body mass in a subject, decreasing adiposity in a subject,decreasing percentage body fat in a subject, and preventing or treatingobesity or a condition related thereto.

F. Methods of Treatment

The invention is drawn inter alia to methods including, but not limitedto, methods of decreasing body mass in a subject, of decreasingadiposity in a subject, of decreasing percentage body fat in a subject,and of preventing or treating obesity or a condition related thereto,comprising administering to a subject in need of said decreasing,preventing or treating with a modulator of the invention. In someembodiments, the modulator is an inverse agonist or antagonist. In someembodiments, the modulator is an inverse agonist. In some embodiments,the modulator is an antagonist. In some embodiments, said modulator isorally active. In some embodiments, said orally active modulator isfurther able to cross the blood-brain barrier. In some embodiments, themodulator is administered to the subject in a pharmaceuticalcomposition. In some embodiments, the modulator is provided to thesubject in a pharmaceutical composition. In some embodiments, themodulator is provided to the subject in a pharmaceutical compositionthat is taken orally. In some embodiments, the subject is a non-humanmammal. In some embodiments, the subject is a mammal. In certainembodiments, the mammal is a mouse, a rat, a non-human primate, or ahuman. In certain preferred embodiments, the subject or mammal is ahuman.

In some embodiments, the subject is in need of having body massdecreased. In some embodiments, the subject is in need of havingpercentage body fat decreased. In some embodiments, the subject is inneed of preventing or treating obesity or a condition related thereto.

In some embodiments, the subject is overweight or obese. In someembodiments, the subject is overweight. In some embodiments, the subjectis obese.

In some embodiments, the obesity comprises weight gain induced by a highfat diet.

In some embodiments, the condition related to obesity is selected fromthe group consisting of hypertension, congestive cardiomyopathy,varicosities, pulmonary embolism, coronary heart disease, stroke,idiopathic intracranial hypertension, meralgia parethetica, dyspnea,obstructive sleep apnea, hypoventilation syndrome, Pickwickian syndrome,asthma, immobility, degenerative osteoarthritis, low back pain, striaedistensae or “stretch marks,” venous stasis of the lower extremities,lymphedema, cellulitis, intertrigo, carbuncles, acanthosis nigricans,skin tags, gastro-esophageal reflux disorder, nonalcoholic fattyliver/steatohepatitis, cholelithiasis, hernias, colon cancer, stressincontinence, obesity-related glomerulopathy, breast and uterine cancer,depression and low self-esteem, impaired quality of life, metabolicsyndrome, insulin resistance, Type 2 diabetes, dyslipidemia,atherosclerosis, hyperandrogenemia in women, polycystic ovariansyndrome, dysmenorrhea, infertility, pregnancy complications, and malehypogonadism. In some embodiments, the condition related to obesity isselected from the group consisting of hypertension, insulin resistance,metabolic syndrome, Type 2 diabetes, dyslipidemia, atherosclerosis,coronary heart disease, and stroke.

The invention also contemplates methods of preventing or treating adisorder ameliorated by increasing body mass including, but not limitedto; cachexia, wasting, AIDS-related weight loss, cancer-related weightloss, anorexia, and bulimia, comprising administering to a subject inneed of said preventing or treating with a modulator of the invention.In some embodiments, the modulator is an agonist or partial agonist. Insome embodiments, the modulator is an agonist. In some embodiments, themodulator is a partial agonist. In some embodiments, said modulator isorally active. In some embodiments, said orally active modulator isfurther able to cross the blood-brain barrier. In some embodiments, themodulator is administered to the subject in a pharmaceuticalcomposition. In some embodiments, the modulator is provided to thesubject in a pharmaceutical composition. In some embodiments, themodulator is provided to the subject in a pharmaceutical compositionthat is taken orally. In some embodiments, the subject is a non-humanmammal. In some embodiments, the subject is a mammal. In certainembodiments, the mammal is a mouse, a rat, a nonhuman primate, or ahuman. In certain preferred embodiments, the subject or mammal is ahuman.

G. Other Utility

Agents that modulate (i.e., increase, decrease, or block) receptorfunctionality of a GPCR of the invention such as a mammalian GPR50 maybe identified by contacting a candidate compound with the GPCR anddetermining the effect of the candidate compound on receptorfunctionality. The selectivity of a compound that modulates thefunctionality of a mammalian GPR50 such as human GPR50 can be evaluatedby comparing its effects on GPR50 to its effects on one or more other Gprotein-coupled receptors. In certain embodiments, a GPR50 selectivemodulator is a GPR50 selective inverse agonist or antagonist having aselectivity for GPR50 over MTNR1A or MTNR1B of at least about 10-fold orof at least about 100-fold. In certain embodiments, a GPR50 selectivemodulator is a GPR50 selective inverse agonist or antagonist having aselectivity for GPR50 over MTNR1A and MTNR1B of at least about 10-foldor of at least about 100-fold. Following identification of compoundsthat modulate GPR50 functionality, such candidate compounds may befurther tested in other assays including, but not limited to, in vivomodels, in order to confirm or quantitate their activity. By way ofillustration and not limitation, the subject invention expresslycontemplates the identification of compounds as modulators of amammalian GPR50 GPCR for use as pharmaceutical agents. Modulators ofGPR50 functionality are therapeutically useful, e.g., in treatment ofdiseases and physiological conditions in which normal or aberrant GPR50functionality is involved.

Agents that are ligands of a GPCR of the invention such as a mammalianGPR50 may be identified by contacting a candidate compound with the GPCRand determining whether the candidate compound binds to the receptor.The selectivity of a compound that binds to a mammalian GPR50 such ashuman GPR50 can be evaluated by comparing its binding to GPR50 to itsbinding to one or more other G protein-coupled receptors. Ligands thatare modulators of GPR50 receptor functionality are therapeuticallyuseful in treatment of diseases and physiological conditions in whichnormal or aberrant GPR50 functionality is involved.

In other embodiments, agents that are modulators (e.g. increase ordecrease) of body mass, adiposity or percentage of body weight in asubject or that are useful as pharmaceutical agents for obesity andconditions related thereto are identified by contacting a candidatecompound with a GPR50 receptor and determining the effect of thecandidate compound on GPR50 receptor expression. In some embodiments,the agent reduces expression of GPR50 receptor in a cell. In someembodiments, the agent reduces expression of GPR50 receptor in aneuronal cell. In some embodiments, the agent reduces expression ofGPR40 receptor in a human neuronal cell. In some embodiments, the GPR50receptor is endogenously expressed by the cell or neuronal cell. In someembodiments, a level of GPR50 receptor expression is measured usinganti-GPR50 receptor antibody. Those of skill in the art are creditedwith the ability to produce antibody to human, rat or mouse GPR50receptor that may be used to measure a level of GPR50 expression in acell. In some embodiments, a level of GPR50 receptor expression ismeasured using radiolableled ligand specific for GPR50 receptor (seeinfra). In some embodiments, a level of GPR50 receptor expression ismeasured by Northern blot or RT-PCR.

The present invention also relates to a method of identifying whether acandidate compound is an agent that reduces expression of GPR50 receptorin a cell, said method comprising the steps of:

-   -   (a) contacting or not contacting a plurality of cells comprising        GPR50 receptor with a candidate compound;    -   (b) measuring the level of GPR50 receptor expression in the        cells contacted with the candidate compound and the level of        GPR50 receptor expression in the cells not contacted with the        candidate compound; and    -   (c) comparing the level of GPR50 receptor expression in the        cells contacted with the candidate compound with the level of        GPR50 receptor expression in the cells not contacted with the        candidate compound; wherein a reduction in the level of GPR50        receptor expression in the cells contacted with the candidate        compound compared with the level of GPR50 receptor expression in        the cells not contacted with the candidate compound is        indicative of the candidate compound being an agent that reduces        expression of GPR50 receptor in a cell.

The present invention also relates to a method of identifying acandidate compound as a pharmaceutical agent for obesity or a conditionrelated thereto, said method comprising the steps of:

-   -   (a) contacting or not contacting a plurality of cells comprising        GPR50 receptor with a candidate compound;    -   (b) measuring the level of GPR50 receptor expression in the        cells contacted with the candidate compound and the level of        GPR50 receptor expression in the cells not contacted with the        candidate compound; and    -   (c) comparing the level of GPR50 receptor expression in the        cells contacted with the candidate compound with the level of        GPR50 receptor expression in the cells not contacted with the        candidate compound; wherein a reduction in the level of GPR50        receptor expression in the cells contacted with the candidate        compound compared with the level of GPR50 receptor expression in        the cells not contacted with the candidate compound is        indicative of the candidate compound being a pharmaceutical        agent for obesity or a condition related thereto.

In certain embodiments, the pharmaceutical agent for obesity or acondition related thereto is a compound for preventing or treatingobesity or a condition related thereto.

In some embodiments, said method of identifying whether a candidatecompound is an agent that reduces expression of GPR50 receptor in a cellis an in vitro method.

In some embodiments, said plurality of cells contacted or not contactedwith the candidate compound in step (a) are cultured for at least about1 hour, at least about 2 hours, at least about 4 hours, at least about 8hours, at least about 16 hours, at least about 24 hours, at least about36 hours or at least about 48 hours before the level of GPR50 receptorexpression in said cells is measured in step (b).

The present invention relates to said agent that reduces GPR50expression in a cell (e.g., a neuronal cell), to a compositioncomprising said agent (e.g., a pharmaceutical composition), and tomethods of using said composition (e.g., for the prevention of ortreatment of obesity or a condition related thereto), wherein thecompound is a small molecule. The present invention relates to saidagent that reduces GPR50 expression in a cell (e.g., a neuronal cell),to a composition comprising said agent (e.g., a pharmaceuticalcomposition), and to methods of using said composition (e.g., for theprevention of or treatment of obesity or a condition related thereto),wherein the compound is antisense nucleic acid (e.g., antisense RNA).The present invention relates to said agent that reduces GPR50expression in a cell (e.g., a neuronal cell), to a compositioncomprising said agent (e.g., a pharmaceutical composition), and tomethods of using said composition (e.g., for the prevention of ortreatment of obesity or a condition related thereto), wherein thecompound is a small interfering RNA (siRNA) or short hairpin RNA (shRNA)molecule comprising a nucleotide sequence derived from the nucleotidesequence of a GPR50 receptor-encoding gene according to standardprocedures. As will be known to the skilled artisan, siRNA, shRNA andantisense RNA are generally capable of modulating expression of a targetgene [see, e.g., Holmlund J T, Ann NY Acad Sci (2003) 1002:244-251; andDevroe et al, Expert Opin Biol Ther (2004) 4:319-327; the disclosure ofeach of which is hereby incorporated by reference in its entirety].

The present invention also relates to radioisotope-labeled versions ofcompounds of the invention identified as modulators or ligands of a GPCRof the invention such as a mammalian GPR50 that would be useful not onlyin radio-imaging but also in assays, both in vitro and in vivo, forlocalizing and quantitating GPR50 in tissue samples, including human,and for identifying GPR50 ligands in methods relating to inhibition ofbinding of a radioisotope-labeled compound such as a known ligand ofGPR50. It is a further object of this invention to develop novel assaysrelating to a GPCR of the invention such as a mammalian GPR50, such ashuman GPR50, which comprise such radioisotope-labeled compounds. By wayof illustration and not limitation, it is envisioned that elevated brainGPR50 above a normal range visualized by radio-imaging identifies asubject at risk for obesity or a condition related thereto. In someembodiments, the brain GPR50 is hypothalamic GPR50. In some embodiments,the brain GPR50 is pituitary GPR50. In some embodiments, the subject isa human.

The present invention also relates a method of radio-imaging comprisingadministering to a mammal in need of said radio-imaging a radiolabeledcompound that is a modulator or a ligand of the mammalian GPR50receptor. In one aspect, the ligand of the mammalian GPR50 receptor isnot a modulator of the mammalian GPR50 receptor. In some embodiments,the mammal is a human. In some embodiments, the method of radio-imagingis for identifying whether the mammal is at risk for or progressingtoward obesity or a condition related thereto, wherein a level of brainGPR50 in the mammal above the normal range is indicative of the mammalbeing at risk for or progressing toward obesity or a condition relatedthereto. In some embodiments, the method of radio-imaging is foridentifying the mammal for prevention or treatment of obesity or acondition related thereto with an inverse agonist or an antagonist ofthe mammalian GPR50 or with an agent that decreases GPR50 expression ina cell or with a pharmaceutical composition comprising the inverseagonist or the antagonist or the agent and a pharmaceutically acceptablecarrier, wherein a level of brain GPR50 in the mammal above a normalrange identifying the mammal for prevention or treatment of obesity or acondition related thereto with the inverse agonist or the antagonist ofthe mammalian GPR50 or with the agent that decreases GPR50 expression ina cell or with the pharmaceutical composition comprising the inverseagonist or the antagonist or the agent and a pharmaceutically acceptablecarrier. In some embodiments, the brain GPR50 is hypothalamic GPR50. Insome embodiments, the brain GPR50 is pituitary GPR50.

The present invention embraces radioisotope-labeled versions ofcompounds of the invention identified as modulators or ligands of a GPCRof the invention such as a mammalian GPR50, such as human GPR50.

The present invention also relates to radioisotope-labeled versions oftest ligands that are useful for detecting a ligand bound to a GPCR ofthe invention such as a mammalian GPR50, such as human GPR50. In someembodiments, the present invention expressly contemplates a library ofsaid radiolabeled test ligands useful for detecting a ligand bound to aGPCR of the invention such as a mammalian GPR50, such as human GPR50. Incertain embodiments, said library comprises at least about 10, at leastabout 10², at least about 10³, at least about 10⁵, or at least about 10⁶said radiolabeled test compounds. It is a further object of thisinvention to develop novel assays relating to a GPCR of the inventionsuch as a mammalian GPR50, such as human GPR50, which comprise suchradioisotope-labeled test ligands.

In some embodiments, a radioisotope-labeled version of a compound isidentical to the compound, but for the fact that one or more atoms arereplaced or substituted by an atom having an atomic mass or mass numberdifferent from the atomic mass or mass number typically found in nature(i.e., naturally occurring). Suitable radionuclides that may beincorporated in compounds of the present invention include but are notlimited to ²H (deuterium), ³H (tritium), ¹¹C, ¹³C, ¹⁴C, ¹³N, ¹⁵N, ¹⁵O,¹⁷O, ¹⁸O, ¹⁸F, ³⁵S, ³⁶Cl, ⁸²Br, ⁷⁵Br, ⁷⁶Br, ⁷⁷Br, ¹²³I, ¹²⁴I, ¹²⁵I and¹³¹I. The radionuclide that is incorporated in the instant radio-labeledcompound will depend on the specific application of that radio-labeledcompound. For example, for in vitro GPR50 receptor labeling andcompetition assays, compounds that incorporate ³H, ¹⁴C, ⁸²Br, ¹²⁵I,¹³¹I, ³⁵S or will generally be most useful. For radio-imagingapplications ¹¹C, ¹⁸F, ¹²⁵I, ¹²³I, ¹²⁴I, ¹³¹I, ⁷⁵Br, ⁷⁶Br or ⁷⁷Br willgenerally be most useful. In some embodiments, the radionuclide isselected from the group consisting of ³H, ¹¹C, ¹⁸F, ¹⁴C, ¹²⁵I, ¹²⁴I,¹³¹I, ³⁵S and ⁸²Br.

Synthetic methods for incorporating radio-isotopes into organiccompounds are applicable to compounds of the invention and are wellknown in the art. These synthetic methods, for example, incorporatingactivity levels of tritium into target molecules, are as follows:

A. Catalytic Reduction with Tritium Gas—This procedure normally yieldshigh specific activity products and requires halogenated or unsaturatedprecursors.

B. Reduction with Sodium Borohydride [³H]—This procedure is ratherinexpensive and requires precursors containing reducible functionalgroups such as aldehydes, ketones, lactones, esters, and the like.

C. Reduction with Lithium Aluminum Hydride [³H]—This procedure offersproducts at almost theoretical specific activities. It also requiresprecursors containing reducible functional groups such as aldehydes,ketones, lactones, esters, and the like.

D. Tritium Gas Exposure Labeling—This procedure involves exposingprecursors containing exchangeable protons to tritium gas in thepresence of a suitable catalyst.

E. N-Methylation using Methyl Iodide [3H]—This procedure is usuallyemployed to prepare O-methyl or N-methyl (³H) products by treatingappropriate precursors with high specific activity methyl iodide (³H).This method in general allows for higher specific activity, such as forexample, about 70-90 Ci/mmol.

Synthetic methods for incorporating activity levels of ¹²⁵I into targetmolecules include:

A. Sandmeyer and like reactions—This procedure transforms an aryl orheteroaryl amine into a diazonium salt, such as a tetrafluoroboratesalt, and subsequently to ¹²⁵I labeled compound using Na¹²⁵I. Arepresented procedure was reported by Zhu, D.-G. and co-workers in J.Org. Chem. 2002, 67, 943-948.

B. Ortho ¹²⁵Iodination of phenols—This procedure allows for theincorporation of ¹²⁵I at the ortho position of a phenol as reported byCollier, T. L. and co-workers in J. Labeled Compd Radiopharm. 1999, 42,S264-S266.

C. Aryl and heteroaryl bromide exchange with ¹²⁵I—This method isgenerally a two step process. The first step is the conversion of thearyl or heteroaryl bromide to the corresponding tri-alkyltinintermediate using for example, a Pd catalyzed reaction [i.e. Pd(Ph₃P)₄]or through an aryl or heteroaryl lithium, in the presence of atri-alkyltinhalide or hexaalkylditin [e.g., (CH₃)₃SnSn(CH₃)₃]. Arepresented procedure was reported by Bas, M.-D. and co-workers in J.Labeled Compd Radiopharm. 2001, 44, S280-S282.

In some embodiments, a radioisotope-labeled version of a compound isidentical to the compound, but for the addition of one or moresubstituents comprising a radionuclide. In some further embodiments, thecompound is a polypeptide. In some further embodiments, the compound isan antibody or an antigen-binding fragment thereof. In some furtherembodiments, said antibody is monoclonal. Suitable said radionuclideincludes but is not limited to ²H (deuterium), ³H (tritium), ¹¹C, ¹³C,¹⁴C, ¹³N, ¹⁵N, ¹⁵O, ¹⁷O, ¹⁸O, ¹⁸F, ³⁵S, ³⁶Cl, ⁸²Br, ⁷⁵Br, ⁷⁶Br, ⁷⁷Br,¹²³I, ¹²⁴I, ¹²⁵I and ¹³¹I. The radionuclide that is incorporated in theinstant radio-labeled compound will depend on the specific applicationof that radio-labeled compound. For example, for in vitro GPR50 receptorlabeling and competition assays, compounds that incorporate ³H, ¹⁴C,⁸²Br, ¹²⁵I, ¹³¹I, ³⁵S or will generally be most useful. Forradio-imaging applications ¹¹C, ¹⁸F, ¹²⁵I, ¹²³I, ¹²⁴I, ¹³¹I, ⁷⁵Br, ⁷⁶Bror ⁷⁷Br will generally be most useful. In some embodiments, theradionuclide is selected from the group consisting of ³H, ¹¹C, ¹⁸F, ¹⁴C,¹²⁵I, ¹²⁴I, ¹³¹I, ³⁵S and ⁸²Br.

Methods for adding one or more substituents comprising a radionuclideare within the purview of the skilled artisan and include, but are notlimited to, addition of radioisotopic iodine by enzymatic method[Marchalonic J J, Biochemical Journal (1969) 113:299-305; Thorell J Iand Johansson B G, Biochimica et Biophysica Acta (1969) 251:363-9; thedisclosure of each of which is herein incorporated by reference in itsentirety] and or by Chloramine-T/Iodogen/Iodobead methods [Hunter W Mand Greenwood F C, Nature (1962) 194:495-6; Greenwood F C et al.,Biochemical Journal (1963) 89:114-23; the disclosure of each of which isherein incorporated by reference in its entirety].

Other uses of the disclosed receptors and methods will become apparentto those in the art based upon, inter alia, a review of this patentdocument.

EXAMPLES

The following examples are presented for purposes of elucidation, andnot limitation, of the present invention. While specific nucleic acidand amino acid sequences are disclosed herein, those of ordinary skillin the art are credited with the ability to make minor modifications tothese sequences while achieving the same or substantially similarresults reported below. Such modified approaches are considered withinthe purview of this disclosure. Without further elaboration, it isbelieved that one skilled in the art can, using the precedingdescription, practice the present invention to its fullest extent. Thefollowing detailed examples are to be construed as merely illustrative,and not limitations of the preceding disclosure in any way whatsoever.Those skilled in the art will promptly recognize appropriate variationsfrom the procedures.

Recombinant DNA techniques relating to the subject matter of the presentinvention and well known to those of ordinary skill in the art can befound, e.g, in Maniatis T et al., Molecular Cloning: A Laboratory Manual(1989) Cold Spring Harbor Laboratory; U.S. Pat. No. 6,399,373; and PCTApplication Number PCT/IB02/01461 published as WO 02/066505 on 29 Aug.2002; the disclosure of each of which is herein incorporated byreference in its entirety.

Example 1 Full-Length Cloning of Endogenous Human GPR50

Polynucleotide encoding endogenous human GPR50 was cloned by reversetranscription polymerase chain reaction (RT-PCR) using the GPR50specific primers 5′-GGAAAGCTTAACGATCCCCAGGAGCAACAT-3′ (SEQ ID NO: 9;sense with HindIII site and the last two nucleotides being part of theinitiation codon) and 5′-CTGGGATCCTACGAGAGCATTTTTCACACAG-3′ (SEQ ID NO:10; antisense with BamHI site, TCA as antisense of stop codon) and humanpituitary cDNA (Clontech) as template. Cloned pfu DNA polymerase(Stratagene) was used for amplification by the following cycle with step2 to step 4 repeated 25 times:94° C., 3 minutes; 94° C., 1 minute; 62° C., 1 minute; 72° C., 3 minute;72° C., 10 minutes.

A 1.9 Kb PCR fragment of predicted size was isolated, digested withHindIII and BamHI, and cloned into the pCMV expression vector andsequenced using the T7 DNA sequenase kit (Amersham). See, SEQ ID NO: 1for a first nucleic acid sequence obtained in this manner and SEQ IDNO:2 for the deduced amino acid sequence. See, SEQ ID NO:3 for a secondnucleic acid sequence obtained in this manner and SEQ ID NO:4 for thededuced amino acid sequence.

Example 2 Receptor Expression

Although a variety of cells are available to the art for the expressionof proteins, it is most preferred that mammalian cells or melanophoresbe utilized. The primary reason for this is predicated uponpracticalities, i.e., utilization of, e.g. yeast cells for theexpression of a GPCR, while possible, introduces into the protocol anon-mammalian cell which may not (indeed, in the case of yeast, doesnot) include the receptor-coupling, genetic-mechanism and secretarypathways that have evolved for mammalian systems—thus, results obtainedin non-mammalian cells, while of potential use, are not as preferred asthat obtained from mammalian cells or melanophores. Of the mammaliancells, CHO, COS-7, MCB3901, 293 and 293T cells are particularlypreferred, although the specific mammalian cell utilized can bepredicated upon the particular needs of the artisan. See infra asrelates to melanophores, including Example 9.

a. Transient Transfection

On day one, 4×10⁶ 293 cells per 10 cm dish are plated out. On day two,two reaction tubes are prepared (the proportions to follow for each tubeare per plate): tube A is prepared by mixing 4 μg DNA (e.g., pCMVvector; pCMV vector comprising polynucleotide encoding a GPCR of theinvention, etc.) in 0.5 ml serum free DMEM (Gibco BRL); tube B isprepared by mixing 24 μl lipofectamine (Gibco BRL) in 0.5 ml serum freeDMEM. Tubes A and B are admixed by inversions (several times), followedby incubation at room temperature for 30-45 min. The admixture isreferred to as the “transfection mixture”. Plated 293 cells are washedwith 1×PBS, followed by addition of 5 ml serum free DMEM. 1 ml of thetransfection mixture is added to the cells, followed by incubation for 4hrs at 37° C./5% CO₂. The transfection mixture is removed by aspiration,followed by the addition of 10 ml of DMEM/10% Fetal Bovine Serum. Cellsare incubated at 37° C./5% CO₂. After 48 hr incubation, cells areharvested and utilized for analysis.

b. Stable Cell Lines

Approximately 12×10⁶ 293 cells are plated on a 15 cm tissue cultureplate. Grown in DME High Glucose Medium containing ten percent fetalbovine serum and one percent sodium pyruvate, L-glutamine, andantibiotics. Twenty-four hours following plating of 293 cells (or to˜80% confluency), the cells are transfected using 12 μg of DNA (e.g.,pCMV-neo^(r) vector comprising polynucleotide encoding a GPCR of theinvention). The 12 μg of DNA is combined with 60 μl of lipofectamine and2 ml of DME High Glucose Medium without serum. The medium is aspiratedfrom the plates and the cells are washed once with medium without serum.The DNA, lipofectamine, and medium mixture are added to the plate alongwith 10 ml of medium without serum. Following incubation at 37° C. forfour to five hours, the medium is aspirated and 25 ml of mediumcontaining serum is added. Twenty-four hours following transfection, themedium is aspirated again, and fresh medium with serum is added.Forty-eight hours following transfection, the medium is aspirated andmedium with serum is added containing geneticin (G418 drug) at a finalconcentration of 500 μg/ml. The transfected cells now undergo selectionfor positively transfected cells containing the G418 resistance gene.The medium is replaced every four to five days as selection occurs.During selection, cells are grown to create stable pools, or split forstable clonal selection.

Example 3 Assays for Determination of GPCR Activation (e.g., ScreeningAssays)

A variety of approaches are available for assessing activation of a GPCRof interest, or “target” GPCR. The following are illustrative; those ofordinary skill in the art are credited with the ability to determinethose techniques that are preferentially beneficial for the needs of theartisan.

1. Membrane Binding Assays: [³⁵S]GTPγS Assay

When a G protein-coupled receptor is in its active state, either as aresult of ligand binding or constitutive activation, the receptorcouples to a G protein and stimulates the release of GDP and subsequentbinding of GTP to the G protein. The alpha subunit of the Gprotein-receptor complex acts as a GTPase and slowly hydrolyzes the GTPto GDP, at which point the receptor normally is deactivated. Activatedreceptors continue to exchange GDP for GTP. The non-hydrolyzable GTPanalog, [³⁵S]GTPγS, can be utilized to demonstrate enhanced binding of[³⁵S]GTPγS to membranes expressing activated receptors. The advantage ofusing [³⁵S]GTPγS binding to measure activation is that: (a) it isgenerically applicable to all G protein-coupled receptors; (b) it isproximal at the membrane surface making it less likely to pick-upmolecules which affect the intracellular cascade.

The assay utilizes the ability of G protein coupled receptors tostimulate [³⁵S]GTPγS binding to membranes expressing the relevantreceptors. The assay can, therefore, be used to screen candidatecompounds as modulators of GPCRs. The assay is generic and hasapplication to drug discovery at all G protein-coupled receptors.

The [³⁵S]GTPγS assay is incubated in 20 mM HEPES and between 1 and about20 mM MgCl₂ (this amount can be adjusted for optimization of results,although 20 mM is preferred) pH 7.4, binding buffer with between about0.3 and about 1.2 nM [³⁵S]GTPγS (this amount can be adjusted foroptimization of results, although 1.2 is preferred) and 12.5 to 75 μgmembrane protein (e.g. 293 cells expressing a GPCR of the invention;this amount can be adjusted for optimization) and 10 μM GDP (this amountcan be changed for optimization) for 1 hour. Wheatgerm agglutinin beads(25 μl; Amersham) are then added and the mixture incubated for another30 minutes at room temperature. The tubes are then centrifuged at 1500×gfor 5 minutes at room temperature and then counted in a scintillationcounter.

2. Adenylyl Cyclase

A Flash Plate™ Adenylyl Cyclase kit (New England Nuclear; Cat. No.SMP004A) designed for cell-based assays can be modified for use withcrude plasma membranes. The Flash Plate wells can contain a scintillantcoating which also contains a specific antibody recognizing cAMP. ThecAMP generated in the wells can be quantitated by a direct competitionfor binding of radioactive cAMP tracer to the cAMP antibody. Thefollowing serves as a brief protocol for the measurement of changes incAMP levels in whole cells that express the receptors.

Transfected cells are harvested approximately twenty-four to forty-eighthours after transient transfection. Media is carefully aspirated off anddiscarded. 10 ml of PBS is gently added to each dish of cells followedby careful aspiration. 1 ml of Sigma cell dissociation buffer and 3 mlof PBS are added to each plate. Cells are pipetted off the plate and thecell suspension is collected into a 50 ml conical centrifuge tube. Cellsare then centrifuged at room temperature at 1,100 rpm for 5 min. Thecell pellet is carefully re-suspended into an appropriate volume of PBS(about 3 ml/plate). The cells are then counted using a hemocytometer andadditional PBS is added to give the appropriate number of cells (with afinal volume of about 50 μl/well).

cAMP standards and Detection Buffer (comprising 1 μCi of tracer [¹²⁵I]cAMP (50 μl) to 11 ml Detection Buffer) is prepared and maintained inaccordance with the manufacturer's instructions. Assay Buffer isprepared fresh for screening and contains 50 μl of Stimulation Buffer, 3ul of test compound (12 μM final assay concentration) and 50 μl cells.Assay Buffer is stored on ice until utilized. The assay, preferablycarried out e.g. in a 96-well plate, is initiated by addition of 50 μlof cAMP standards to appropriate wells followed by addition of 50 ul ofPBS to wells H-11 and H12. 50 μl of Stimulation Buffer is added to allwells. DMSO (or selected candidate compounds) is added to appropriatewells using a pin tool capable of dispensing 3 μl of compound solution,with a final assay concentration of 12 μM test compound and 100 μl totalassay volume. The cells are then added to the wells and incubated for 60min at room temperature. 100 μl of Detection Mix containing tracer cAMPis then added to the wells. Plates are then incubated additional 2 hoursfollowed by counting in a Wallac MicroBeta scintillation counter. Valuesof cAMP/well are then extrapolated from a standard cAMP curve which iscontained within each assay plate.

3. Cell-Based cAMP Assay for Gi-Coupled Target GPCRs

TSHR is a Gs coupled GPCR that causes the accumulation of cAMP uponactivation. TSHR will be constitutively activated by mutating amino acidresidue 623 (i.e., changing an alanine residue to an isoleucineresidue). A Gi coupled receptor is expected to inhibit adenylyl cyclase,and, therefore, decrease the level of cAMP production, which can makeassessment of cAMP levels challenging. An effective technique formeasuring the decrease in production of cAMP as an indication ofactivation of a Gi coupled receptor can be accomplished byco-transfecting, most preferably, non-endogenous, constitutivelyactivated TSHR (TSHR-A623I) (or an endogenous, constitutively active Gscoupled receptor) as a “signal enhancer” with a Gi coupled Target GPCRto establish a baseline level of cAMP. The Gi coupled receptor isco-transfected with the signal enhancer, and it is this material thatcan be used for screening. Such an approach can be utilized toeffectively generate a signal when a cAMP assay is used. In someembodiments, this approach is preferably used in the identification ofcandidate compounds against Gi coupled receptors. It is noted that for aGi coupled GPCR, when this approach is used, an inverse agonist of theTarget GPCR will increase the cAMP signal and an agonist will decreasethe cAMP signal.

On day one, 4×10⁶ 293 cells per 10 cm dish will be plated out. On daytwo, two reaction tubes will be prepared (the proportions to follow foreach tube are per plate): tube A will be prepared by mixing 2 μg DNA ofeach receptor transfected into the mammalian cells, for a total of 4 μgDNA (e.g., pCMV vector; pCMV vector with mutated THSR (TSHR-A623I);TSHR-A623I and the Target GPCR, etc.) in 0.5 ml serum free DMEM (IrvineScientific, Irvine, Calif.); tube B will be prepared by mixing 24 μllipofectamine (Gibco BRL) in 0.5 ml serum free DMEM. Tubes A and B willthen be admixed by inversions (several times), followed by incubation atroom temperature for 30-45 min. The admixture is referred to as the“transfection mixture”. Plated 293 cells will be washed with 1×PBS,followed by addition of 5 ml serum free DMEM. 1.0 ml of the transfectionmixture will then be added to the cells, followed by incubation for 4hrs at 37° C./5% CO₂. The transfection mixture will then be removed byaspiration, followed by the addition of 10 ml of DMEM/10% Fetal BovineSerum. Cells will then be incubated at 37° C./5% CO₂. Afterapproximately 24-48 hr incubation, cells will then be harvested andutilized for analysis.

A Flash Plate™ Adenylyl Cyclase kit (New England Nuclear; Cat. No.SMP004A) is designed for cell-based assays, but can be modified for usewith crude plasma membranes depending on the need of the skilledartisan. The Flash Plate wells will contain a scintillant coating whichalso contains a specific antibody recognizing cAMP. The cAMP generatedin the wells can be quantitated by a direct competition for binding ofradioactive cAMP tracer to the cAMP antibody. The following serves as abrief protocol for the measurement of changes in cAMP levels in wholecells that express the receptors.

Transfected cells will be harvested approximately twenty-four toforty-eight hours after transient transfection. Media will be carefullyaspirated off and discarded. 10 ml of PBS will be gently added to eachdish of cells followed by careful aspiration. 1 ml of Sigma celldissociation buffer and 3 ml of PBS will be added to each plate. Cellswill be pipetted off the plate and the cell suspension will be collectedinto a 50 ml conical centrifuge tube. Cells will then be centrifuged atroom temperature at 1,100 rpm for 5 min. The cell pellet will becarefully re-suspended into an appropriate volume of PBS (about 3ml/plate). The cells will then be counted using a hemocytometer andadditional PBS is added to give the appropriate number of cells (with afinal volume of about 50 μl/well).

cAMP standards and Detection Buffer (comprising 1 μCi of tracer [¹²⁵I]cAMP (50 μl) to 11 ml Detection Buffer) will be prepared and maintainedin accordance with the manufacturer's instructions. Assay Buffer shouldbe prepared fresh for screening and contained 50 μl of StimulationBuffer, 3 μl of test compound (12 μM final assay concentration) and 50μl cells, Assay Buffer can be stored on ice until utilized. The assaycan be initiated by addition of 50 μl of cAMP standards to appropriatewells followed by addition of 50 μl of PBS to wells H-11 and H12. Fiftyμl of Stimulation Buffer will be added to all wells. Selected compounds(e.g., TSH) will be added to appropriate wells using a pin tool capableof dispensing 3 μl of compound solution, with a final assayconcentration of 12 μM test compound and 100 μl total assay volume. Thecells will then be added to the wells and incubated for 60 min at roomtemperature. 100 μl of Detection Mix containing tracer cAMP will then beadded to the wells. Plates were then incubated additional 2 hoursfollowed by counting in a Wallac MicroBeta scintillation counter. Valuesof cAMP/well will then be extrapolated from a standard cAMP curve whichis contained within each assay plate.

4. Reporter-Based Assays

a. CRE-Luc Reporter Assay (Gs-Associated Receptors)

293 and 293T cells are plated-out on 96 well plates at a density of2×10⁴ cells per well and were transfected using Lipofectamine Reagent(BRL) the following day according to manufacturer instructions. ADNA/lipid mixture is prepared for each 6-well transfection as follows:260 ng of plasmid DNA in 100 μl of DMEM is gently mixed with 2 μl oflipid in 100 μl of DMEM (the 260 ng of plasmid DNA consists of 200 ng ofa 8×CRE-Luc reporter plasmid, 50 ng of pCMV comprising endogenousreceptor or non-endogenous receptor or pCMV alone, and 10 ng of a GPRSexpression plasmid (GPRS in pcDNA3 (Invitrogen)). The 8×CRE-Luc reporterplasmid was prepared as follows: vector SRIF-β-gal was obtained bycloning the rat somatostatin promoter (−71/+51) at BglV-HindIII site inthe pβgal-Basic Vector (Clontech). Eight (8) copies of cAMP responseelement were obtained by PCR from an adenovirus template AdpCF126CCRE8[see, Suzuki et al., Hum Gene Ther (1996) 7:1883-1893; the disclosure ofwhich is herein incorporated by reference in its entirety) and clonedinto the SRIF-β-gal vector at the Kpn-BglV site, resulting in the8×CRE-β-gal reporter vector. The 8×CRE-Luc reporter plasmid wasgenerated by replacing the beta-galactosidase gene in the 8×CRE-β-galreporter vector with the luciferase gene obtained from the pGL3-basicvector (Promega) at the HindIII-BamHI site. Following 30 min. incubationat room temperature, the DNA/lipid mixture is diluted with 400 μl ofDMEM and 100 μl of the diluted mixture is added to each well. 100 μl ofDMEM with 10% FCS are added to each well after a 4 hr incubation in acell culture incubator. The following day the transfected cells arechanged with 200 μl/well of DMEM with 10% FCS. Eight (8) hours later,the wells are changed to 100 μl/well of DMEM without phenol red, afterone wash with PBS. Luciferase activity is measured the next day usingthe LucLite™ reporter gene assay kit (Packard) following manufacturerinstructions and read on a 1450 MicroBeta™ scintillation andluminescence counter (Wallac).

b. AP1 Reporter Assay (Gq-Associated Receptors)

A method to detect Gq stimulation depends on the known property ofGq-dependent phospholipase C to cause the activation of genes containingAP1 elements in their promoter. A Pathdetect™ AP-1 cis-Reporting System(Stratagene, Catalogue #219073) can be utilized following the protocolset forth above with respect to the CREB reporter assay, except that thecomponents of the calcium phosphate precipitate were 410 ng pAP1-Luc, 80ng pCMV-receptor expression plasmid, and 20 ng CMV-SEAP (secretedalkaline phosphatase expression plasmid; alkaline phosphatase activityis measured in the media of transfected cells to control for variationsin transfection efficiency between samples).

c. SRF-LUC Reporter Assay (Gq-Associated Receptors)

One method to detect Gq stimulation depends on the known property ofGq-dependent phospholipase C to cause the activation of genes containingserum response factors in their promoter. A Pathdetect™SRF-Luc-Reporting System (Stratagene) can be utilized to assay for Gqcoupled activity in, e.g., COS7 cells. Cells are transfected with theplasmid components of the system and the indicated expression plasmidencoding endogenous or non-endogenous GPCR using a MammalianTransfection™ Kit (Stratagene, Catalogue #200285) according to themanufacturer's instructions. Briefly, 410 ng SRF-Luc, 80 ngpCMV-receptor expression plasmid and 20 ng CMV-SEAP are combined in acalcium phosphate precipitate as per the manufacturer's instructions.Half of the precipitate is equally distributed over 3 wells in a 96-wellplate, kept on the cells in a serum free media for 24 hours. The last 5hours the cells are incubated with, e.g. 1 μM, test compound. Cells arethen lysed and assayed for luciferase activity using a Luclite™ Kit(Packard, Cat. #6016911) and “Trilux 1450 Microbeta” liquidscintillation and luminescence counter (Wallac) as per themanufacturer's instructions. The data can be analyzed using GraphPadPrism™ 2.0a (GraphPad Software Inc.).

d. Intracellular IP3 Accumulation Assay (Gq-Associated Receptors)

On day 1, cells comprising the receptors (endogenous or non-endogenous)can be plated onto 24 well plates, usually b 1×10 ⁵ cells/well (althoughhis number can be optimized. On day 2 cells can be transfected by firstmixing 0.25 μg DNA in 50 μl serum free DMEM/well and 2 μl lipofectaminein 50 μl serum free DMEM/well. The solutions are gently mixed andincubated for 15-30 min at room temperature. Cells are washed with 0.5ml PBS and 400 μl of serum free media is mined with the transfectionmedia and added to the cells. The cells are then incubated for 3-4 hrsat 37° C./5% CO₂ and then the transfection media is removed and replacedwith 1 ml/well of regular growth media. On day 3 the cells are labeledwith ³H-myo-inositol. Briefly, the media is removed and the cells arewashed with 0.5 ml PBS. Then 0.5 ml inositol-free/serum free media(GIBCO BRL) is added/well with 0.25 μCi of ³H-myo-inositol/well and thecells are incubated for 16-18 hrs o/n at 37° C./5% CO₂. On Day 4 thecells are washed with 0.5 ml PBS and 0.45 ml of assay medium is addedcontaining inositol-free/serum free media 10 μM pargyline 10 mM lithiumchloride or 0.4 ml of assay medium and optionally 50 μl of test compoundto final concentration of 10 μM. The cells are then incubated for 30 minat 37° C. The cells are then washed with 0.5 ml PBS and 200 μl offresh/ice cold stop solution (1M KOH; 18 mM Na-borate; 3.8 mM EDTA) isadded/well. The solution is kept on ice for 5-10 min or until cells werelysed and then neutralized by 200 μl of fresh/ice cold neutralizationsol. (7.5% HCL). The lysate is then transferred into 1.5 ml eppendorftubes and 1 ml of chloroform/methanol (1:2) is added/tube. The solutionis vortexed for 15 sec and the upper phase is applied to a BioradAG1-X8™ anion exchange resin (100-200 mesh). Firstly, the resin iswashed with water at 1:1.25 W/V and 0.9 ml of upper phase is loaded ontothe column. The column is washed with 10 mls of 5 mM myo-inositol and 10ml of 5 mM Na-borate/60 mM Na-formate. The inositol tris phosphates areeluted into scintillation vials containing 10 ml of scintillationcocktail with 2 ml of 0.1 M formic acid/1 M ammonium formate. Thecolumns are regenerated by washing with 10 ml of 0.1 M formic acid/3Mammonium formate and rinsed twice with dd H₂O and stored at 4° C. inwater.

Example 4 Fusion Protein Preparation

a. GPCR:Gs Fusion Construct

The design of the GPCR-G protein fusion construct can be accomplished asfollows: both the 5′ and 3′ ends of the rat G protein Gsα (long form;Itoh, H. et al., 83 PNAS 3776 (1986)) are engineered to include aHindIII (5′-AAGCTT-3′) sequence thereon. Following confirmation of thecorrect sequence (including the flanking HindIII sequences), the entiresequence is shuttled into pcDNA3.1(−) (Invitrogen, cat. no. V795-20) bysubcloning using the HindIII restriction site of that vector. Thecorrect orientation for the Gsα sequence is determined after subcloninginto pcDNA3.1(−). The modified pcDNA3.1(−) containing the rat Gsα geneat HindIII sequence is then verified; this vector is now available as a“universal” Gsα protein vector. The pcDNA3.1(−) vector contains avariety of well-known restriction sites upstream of the HindIII site,thus beneficially providing the ability to insert, upstream of the Gsprotein, the coding sequence of an endogenous, constitutively activeGPCR. This same approach can be utilized to create other “universal” Gprotein vectors, and, of course, other commercially available orproprietary vectors known to the artisan can be utilized—the importantcriteria is that the sequence for the GPCR be upstream and in-frame withthat of the G protein.

b. Gq(6 Amino Acid Deletion)/Gi Fusion Construct

A Gq(del)/Gi fusion construct is a chimeric G protein whereby the firstsix (6) amino acids of the Gq-protein α-subunit (“Gαq”) are deleted andthe last five (5) amino acids at the C-terminal end of Gαq are replacedwith the corresponding amino acids of the Gαi subunit. A Gq(del)/Gifusion construct will force an endogenous Gi coupled receptor to coupleto its non-endogenous G protein, Gq (in the form of Gq(del)/Gi), suchthat the second messenger, for example, inositol triphosphate ordiacylglycerol or Ca²⁺, can be measured in lieu of cAMP production.

The Gq(del)/Gi fusion construct was designed as follows: the N-terminalsix (6) amino acids (amino acids 2 through 7, having the sequence ofTLESIM (SEQ ID NO: 11) of the Gαq-subunit were deleted and theC-terminal five (5) amino acids, having the sequence EYNLV (SEQ ID NO:12) were replaced with the corresponding amino acids of the Gαi Protein,having the sequence DCGLF (SEQ ID NO: 13). This fusion construct wasobtained by PCR using the following primers:5′-gatcaagcttcCATGGCGTGCTGCCTGAGCGAGGAG-3′ (SEQ ID NO: 14) and5′-gateggatcTTAGAACAGGCCGCAGTCCTTCAGGTTCAGCTGCAGGATGGTG-3′ (SEQ ID NO:15) and Plasmid 63313 (ATCC® Number 63313) which contains the mouseGαq-wild-type version with a hemagglutinin tag as a template.Nucleotides in lower case include cloning sites for HindIII/BamHI andspacers.

TaqPlus Precision DNA polymerase (Stratagene) was utilized for theamplification by the following cycles, with steps 2 through 4 repeated35 times: 95° C. for 2 min; 95° C. for 20 sec; 56° C. for 20 sec; 72° C.for 2 min; and 72° C. for 7 min. The PCR product was cloned intopCRII-TOPO vector (Invitrogen) and sequenced using the ABI Big DyeTerminator kit (P. E. Biosystems). Inserts from a TOPO clone containingthe sequence of the fusion construct was shuttled into the expressionvector pcDNA3.1(+) at the HindIII/BamHI site by a 2 step cloningprocess. See, SEQ ID NO: 16 for the nucleic acid sequence and SEQ ID NO:17 for the encoded amino acid sequence of Gq(del)/Gi construct.

Example 5 [³⁵S]GTPγS Assay

1. Membrane Preparation

In some embodiments membranes comprising a Target GPCR and for use inthe identification of candidate compounds as, e.g., inverse agonists,agonists, or antagonists, are preferably prepared as follows:

a. Materials

“Membrane Scrape Buffer” is comprised of 20 mM HEPES and 10 mM EDTA, pH7.4; “Membrane Wash Buffer” is comprised of 20 mM HEPES and 0.1 mM EDTA,pH 7.4; “Binding Buffer” is comprised of 20 mM HEPES, 100 mM NaCl, and10 mM MgCl₂, pH 7.4.

b. Procedure

All materials will be kept on ice throughout the procedure. Firstly, themedia will be aspirated from a confluent monolayer of cells, followed byrinse with 10 ml cold PBS, followed by aspiration. Thereafter, 5 ml ofMembrane Scrape Buffer will be added to scrape cells; this will befollowed by transfer of cellular extract into 50 ml centrifuge tubes(centrifuged at 20,000 rpm for 17 minutes at 4° C.). Thereafter, thesupernatant will be aspirated and the pellet will be resuspended in 30ml Membrane Wash Buffer followed by centrifuge at 20,000 rpm for 17minutes at 4° C. The supernatant will then be aspirated and the pelletresuspended in Binding Buffer. This will then be homogenized using aBrinkman Polytron™ homogenizer (15-20 second bursts until the allmaterial is in suspension). This is referred to herein as “MembraneProtein”.

2. Bradford Protein Assay

Following the homogenization, protein concentration of the membraneswill be determined using the Bradford Protein Assay (protein can bediluted to about 1.5 mg/ml, aliquoted and frozen (−80° C.) for lateruse; when frozen, protocol for use will be as follows: on the day of theassay, frozen Membrane Protein is thawed at room temperature, followedby vortex and then homogenized with a Polytron at about 12×1,000 rpm forabout 5-10 seconds; it is noted that for multiple preparations, thehomogenizer should be thoroughly cleaned between homogenization ofdifferent preparations).

a. Materials

Binding Buffer (as per above); Bradford Dye Reagent; Bradford ProteinStandard will be utilized, following manufacturer instructions (Biorad,cat. no. 500-0006).

b. Procedure

Duplicate tubes will be prepared, one including the membrane, and one asa control “blank”. Each contained 800 μl Binding Buffer. Thereafter, 10μl of Bradford Protein Standard (1 mg/ml) will be added to each tube,and 10 μl of membrane Protein will then be added to just one tube (notthe blank). Thereafter, 200 μl of Bradford Dye Reagent will be added toeach tube, followed by vortex of each. After five (5) minutes, the tubeswill be re-vortexed and the material therein will be transferred tocuvettes. The cuvettes will then be read using a CECIL 3041spectrophotometer, at wavelength 595 nm.

3. Identification Assay

a. Materials

GDP Buffer consists of 37.5 ml Binding Buffer and 2 mg GDP (Sigma, cat.no. G-7127), followed by a series of dilutions in Binding Buffer toobtain 0.2 μM GDP (final concentration of GDP in each well was 0.1 μMGDP); each well comprising a candidate compound, has a final volume of200 μl consisting of 100 μl GDP Buffer (final concentration, 0.1 μMGDP), 50 μl Membrane Protein in Binding Buffer, and 50 μl [³⁵S]GTPγS(0.6 nM) in Binding Buffer (2.5 μl [³⁵S]GTPγS per 10 ml Binding Buffer).

b. Procedure

Candidate compounds will be preferably screened using a 96-well plateformat (these can be frozen at −80° C.). Membrane Protein (or membraneswith expression vector excluding the Target GPCR, as control), will behomogenized briefly until in suspension. Protein concentration will thenbe determined using the Bradford Protein Assay set forth above. MembraneProtein (and control) will then be diluted to 0.25 mg/ml in BindingBuffer (final assay concentration, 12.5 μg/well). Thereafter, 100 μl GDPBuffer is added to each well of a Wallac Scintistrip™ (Wallac). A 5 ulpin-tool will then be used to transfer 5 μl of a candidate compound intosuch well (i.e., 5 μl in total assay volume of 200 μl is a 1:40 ratiosuch that the final screening concentration of the candidate compound is10 μM). Again, to avoid contamination, after each transfer step the pintool should be rinsed in three reservoirs comprising water (1×), ethanol(1×) and water (2×)—excess liquid should be shaken from the tool aftereach rinse and dried with paper and kimwipes. Thereafter, 50 μl ofMembrane Protein will be added to each well (a control well comprisingmembranes without the Target GPCR was also utilized), and pre-incubatedfor 5-10 minutes at room temperature. Thereafter, 50 μl of [³⁵S]GTPγS(0.6 nM) in Binding Buffer will be added to each well, followed byincubation on a shaker for 60 minutes at room temperature (again, inthis example, plates were covered with foil). The assay will then bestopped by spinning of the plates at 4000 RPM for 15 minutes at 22° C.The plates will then be aspirated with an 8 channel manifold and sealedwith plate covers. The plates will then be read on a Wallac 1450 usingsetting “Prot. #37” (as per manufacturer's instructions).

Example 6 Cyclic AMP Assay

Another assay approach for identifying candidate compounds as, e.g.,inverse agonists, agonists, or antagonists, is accomplished by utilizinga cyclase-based assay. In addition to so identifying candidatecompounds, this assay approach can be utilized as an independentapproach to provide confirmation of the results from the [³⁵S]GTPγSapproach as set forth in Example 5, supra.

A modified Flash Plate™ Adenylyl Cyclase kit (New England Nuclear; Cat.No. SMP004A) is preferably utilized for identification of candidatecompounds as modulators of a Target GPCR in accordance with thefollowing protocol.

Cells transfected with the Target GPCR are harvested approximately threedays after transfection. Membranes are prepared by homogenization ofsuspended cells in buffer containing 20 mM HEPES, pH 7.4 and 10 mMMgCl₂. Homogenization is performed on ice using a Brinkman Polytron™ forapproximately 10 seconds. The resulting homogenate is centrifuged at49,000×g for 15 minutes at 4° C. The resulting pellet is thenresuspended in buffer containing 20 mM HEPES, pH 7.4 and 0.1 mM EDTA,homogenized for 10 seconds, followed by centrifugation at 49,000×g for15 minutes at 4° C. The resulting pellet is then stored at −80° C. untilutilized. On the day of direct identification screening, the membranepellet is slowly thawed at room temperature, resuspended in buffercontaining 20mM HEPES, pH 7.4 and 10 mM MgCl₂, to yield a final proteinconcentration of 0.60 mg/ml (the resuspended membranes are placed on iceuntil use).

cAMP standards and Detection Buffer (comprising 2 μCi of tracer{[¹²⁵I]cAMP (100 μl) to 11 ml Detection Buffer] are prepared andmaintained in accordance with the manufacturer's instructions. AssayBuffer is prepared fresh for screening and contains 20 mM HEPES, pH 7.4,10 mM MgCl₂, 20 mM phospocreatine (Sigma), 0.1 units/ml creatinephospholdinase (Sigma), 50 μM GTP (Sigma), and 0.2 mM ATP (Sigma); AssayBuffer is then stored on ice until utilized.

Candidate compounds are added, preferably, to e.g. 96-well plate wells(3 μl/well; 12 μM final assay concentration), together with 40 μlMembrane Protein (30 μg/well) and 50 μl of Assay Buffer. This admixturewas then incubated for 30 minutes at room temperature, with gentleshaking.

Following the incubation, 100 μl of Detection Buffer is added to eachwell, followed by incubation for 2-24 hours. Plates are then counted ina Wallac MicroBeta™ plate reader using “Prot. #31” (as permanufacturer's instructions).

By way of example and not limitation, an illustrative screening assayplate (96 well format) result obtained is presented in FIG. 1. Each barrepresents the result for a compound that differs in each well, the“Target GPCR” being a Gsα Fusion Protein construct of an endogenous,constitutively active Gs-coupled GPCR unrelated to GPR50. The resultspresented in FIG. 1 also provide standard deviations based upon the meanresults of each plate (“m”) and the mean plus two arbitrary preferencefor selection of inverse agonists as “leads” from the primary screeninvolves selection of candidate compounds that that reduce the per centresponse by at least the mean plate response, minus two standarddeviations. Conversely, an arbitrary preference for selection ofagonists as “leads” from the primary screen involves selection ofcandidate compounds that increase the per cent response by at least themean plate response, plus the two standard deviations. Based upon theseselection processes, the candidate compounds in the following wells weredirectly identified as putative inverse agonist (Compound A) and agonist(Compound B) to said endogenous GPCR in wells A2 and G9, respectively.See, FIG. 1. It is noted for clarity: these compounds have been directlyidentified without any knowledge of the endogenous ligand for this GPCR.By focusing on assay techniques that are based upon receptor function,and not compound binding affinity, it is possible to ascertain compoundsthat are able to reduce the functional activity of this receptor(Compound A) as well as increase the functional activity of the receptor(Compound B).

Example 7 Fluorometric Imaging Plate Reader (FLIPR) Assay for theMeasurement of Intracellular Calcium Concentration

Target Receptor (experimental) and pCMV (negative control) stablytransfected cells from respective clonal lines are seeded intopoly-D-lysine pretreated 96-well plates (Becton-Dickinson, #356640) at5.5×10⁴ cells/well with complete culture medium (DMEM with 10% FBS, 2 mML-glutamine, 1 mM sodium pyruvate) for assay the next day. To prepareFluo4-AM (Molecular Probe, #F14202) incubation buffer stock, 1 mgFluo4-AM is dissolved in 467 μl DMSO and 467 μl Pluoronic acid(Molecular Probe, #P3000) to give a 1 mM stock solution that can bestored at −20° C. for a month. Fluo4-AM is a fluorescent calciumindicator dye.

Candidate compounds are prepared in wash buffer (1×HBSS/2.5 mMProbenicid/20 mM HEPES at pH 7.4).

At the time of assay, culture medium is removed from the wells and thecells are loaded with 100 μl of 4 μM Fluo4-AM/2.5 mM Probenicid (Sigma,#P8761)/20 mM HEPES/complete medium at pH 7.4. Incubation at 37° C./5%CO₂ is allowed to proceed for 60 min.

After the 1 hr incubation, the Fluo4-AM incubation buffer is removed andthe cells are washed 2× with 100 μl wash buffer. In each well is left100 μl wash buffer. The plate is returned to the incubator at 37° C./5%CO₂ for 60 min.

FLIPR (Fluorometric Imaging Plate Reader; Molecular Device) isprogrammed to add 50 μl candidate compound on the 30th second and torecord transient changes in intracellular calcium concentration ([Ca²⁺])evoked by the candidate compound for another 150 seconds. Totalfluorescence change counts are used to determine agonist activity usingthe FLIPR software. The instrument software normalizes the fluorescentreading to give equivalent initial readings at zero.

By way of illustration and not limitation, the skilled artisan wouldappreciate that a candidate compound can be screened as an antagonist ofthe receptor by assessing its ability to inhibit the transient increasein intracellular ([Ca²⁺]) evoked by subsequent contact with a knownagonist. In some embodiments, the cells comprising Target Receptorfurther comprise Gaα5, Gcα16, or Gq(del)/Gi chimeric G protein.

Although the foregoing provides a FLIPR assay for agonist activity usingstably transfected cells, a person of ordinary skill in the art wouldreadily be able to modify the assay in order to characterize antagonistactivity. The person of ordinary skill in the art would also readilyappreciate that, alternatively, transiently transfected cells could beused.

Example 8 MAP Kinase Assay

MAP kinase (mitogen activated kinase) may be monitored to evaluatereceptor activation. MAP kinase can be detected by several approaches.One approach is based on an evaluation of the phosphorylation state,either unphosphorylated (inactive) or phosphorylated (active). Thephosphorylated protein has a slower mobility in SDS-PAGE and cantherefore be compared with the unstimulated protein using Westernblotting. Alternatively, antibodies specific for the phosphorylatedprotein are available (New England Biolabs) which can be used to detectan increase in the phosphorylated kinase. In either method, cells arestimulated with the test compound and then extracted with Laemmlibuffer. The soluble fraction is applied to an SDS-PAGE gel and proteinsare transferred electrophoretically to nitrocellulose or Immobilin.Immunoreactive bands are detected by standard Western blottingtechnique. Visible or chemiluminescent signals are recorded on film andmay be quantified by densitometry.

Another approach is based on evaluation of the MAP kinase activity via aphosphorylation assay. Cells are stimulated with the test compound and asoluble extract is prepared. The extract is incubated at 30° C. for 10min with gamma-³²P-ATP, an ATP regenerating system, and a specificsubstrate for MAP kinase such as phosphorylated heat and acid stableprotein regulated by insulin, or PHAS-I. The reaction is terminated bythe addition of H₃PO₄ and samples are transferred to ice. An aliquot isspotted onto Whatman P81 chromatography paper, which retains thephosphorylated protein. The chromatography paper is washed and countedfor ³²P is a liquid scintillation counter. Alternatively, the cellextract is incubated with gamma-³²P-ATP, an ATP regenerating system, andbiotinylated myelin basic protein bound by streptavidin to a filtersupport. The myelin basic protein is a substrate for activated MAPkinase. The phosphorylation reaction is carried out for 10 min at 30° C.The extract can then be aspirated through the filter, which retains, thephosphorylated myelin basic protein. The filter is washed and countedfor ³²P by liquid scintillation counting.

Example 9 Melanophore Technology

Melanophores are skin cells found in lower vertebrates. They containpigmented organelles termed melanosomes. Melanophores are able toredistribute these melanosomes along a microtubule network uponG-protein coupled receptor (GPCR) activation. The result of this pigmentmovement is an apparent lightening or darkening of the cells. Inmelanophores, the decreased levels of intracellular cAMP that resultfrom activation of a Gi-coupled receptor cause melanosomes to migrate tothe center of the cell, resulting in a dramatic lightening in color. IfcAMP levels are then raised, following activation of a Gs-coupledreceptor, the melanosomes are re-dispersed and the cells appear darkagain. The increased levels of diacylglycerol that result fromactivation of Gq-coupled receptors can also induce this re-dispersion.In addition, the technology is also suited to the study of certainreceptor tyrosine kinases. The response of the melanophores takes placewithin minutes of receptor activation and results in a simple, robustcolor change. The response can be easily detected using a conventionalabsorbance microplate reader or a modest video imaging system. Unlikeother skin cells, the melanophores derive from the neural crest andappear to express a full complement of signaling proteins. Inparticular, the cells express an extremely wide range of G-proteins andso are able to functionally express almost all GPCRs.

Melanophores can be utilized to identify compounds, including naturalligands, against GPCRs. This method can be conducted by introducing testcells of a pigment cell line capable of dispersing or aggregating theirpigment in response to a specific stimulus and expressing an exogenousclone coding for the GCPR. A stimulant, e.g., melatonin, sets an initialstate of pigment disposition wherein the pigment is aggregated withinthe test cells if activation of the GPCR induces pigment dispersion.However, stimulating the cell with a stimulant to set an initial stateof pigment disposition wherein the pigment is dispersed if activation ofthe GPCR induces pigment aggregation. The test cells are then contactedwith chemical compounds, and it is determined whether the pigmentdisposition in the cells changed from the initial state of pigmentdisposition. Dispersion of pigments cells due to the candidate compound,including but not limited to a ligand, coupling to the GPCR will appeardark on a petri dish, while aggregation of pigments cells will appearlight.

Materials and methods can be followed according to the disclosure ofU.S. Pat. No. 5,462,856 and U.S. Pat. No. 6,051,386. These patentdisclosures are herein incorporated by reference in their entirety.

The cells are plated in e.g. 96-well plates (one receptor per plate). 48hours post-transfection, half of the cells on each plate are treatedwith 10 nM melatonin. Melatonin activates an endogenous Gi-coupledreceptor in the melanophores and causes them to aggregate their pigment.The remaining half of the cells are transferred to serum-free medium0.7× L-15 (Gibco). After one hour, the cells in serum-free media remainin a pigment-dispersed state while the melatonin-treated cells are in apigment-aggregated state. At this point, the cells are treated with adose response of a test/candidate compound. If the plated GPCRs bind tothe test/candidate compound, the melanophores would be expected toundergo a color change in response to the compound. If the receptor wereeither a Gs or Gq coupled receptor, then the melatonin-aggregatedmelanophores would undergo pigment dispersion. In contrast, if thereceptor was a Gi-coupled receptor, then the pigment-dispersed cellswould be expected to undergo a dose-dependent pigment aggregation.

Example 10 Transgenic Mouse/Rat/Pig Comprising a Disruption in a GP50Gene

Mouse

A preferred DNA construct will comprise, from 5′-end to 3′-end: (a) afirst nucleotide sequence that is comprised in the mouse GPR50 genomicsequence; (b) a nucleotide sequence comprising a positive selectionmarker, such as the marker for neomycin resistance (neo); and (c) asecond nucleotide sequence that is comprised in the mouse GPR50 genomicsequence and is located on the genome downstream of the first mouseGPR50 nucleotide sequence (a). Mouse GPR50 genomic sequence will beisolated using methods well known to those of ordinary skill in the art(Maniatis T et al., Molecular Cloning: A Laboratory Manual (1989) ColdSpring Harbor Laboratory; the disclosure of which is hereby incorporatedby reference in its entirety). Probes for said isolation of mouse GPR50genomic sequence will be derived from cDNA encoding a mouse GPR50polypeptide, wherein said cDNA may be obtained using as template mRNAfrom mouse heart, lung, or adipose tissue.

In preferred embodiments, this DNA construct also comprises a negativeselection marker located upstream the nucleotide sequence (a) ordownstream the nucleotide sequence (c). Preferably, the negativeselection marker comprises the thymidine kinase (tk) gene [Thomas etal., Cell (1986) 44:419-28], the hygromycin beta gene [Te Riele et al.,Nature (1990) 348:649-51], the hprt gene [Van der Lugt et al., Gene(1991) 105:263-7; Reid et al., Proc Natl Acad Sci USA (1990)87:4299-4303] or the Diptheria toxin A fragment (Dt-A) gene [Nada etal., Cell (1993) 73:1125-35; Yagi et al., Proc Natl Acad Sci USA (1990)87:9918-9922], which disclosures are hereby incorporated by reference intheir entireties. Preferably, the positive selection marker is locatedwithin a mouse GPR50 exon sequence so as to interrupt the sequenceencoding a mouse GPR50 polypeptide. These replacement vectors aredescribed, for example, by Thomas et al., Cell (1986) 44:419-28; Thomaset al., Cell (1987) 51:503-12; Mansour et al., Nature (1988) 336:348-52;Koller et al., Annu Rev Immunol (1992) 10:705-30; and U.S. Pat. No.5,631,153; which disclosures are hereby incorporated by reference intheir entireties.

The first and second nucleotide sequences (a) and (c) may beindifferently located within a mouse GPR50 regulatory sequence, anintronic sequence, an exon sequence or a sequence containing bothregulatory and/or intronic and/or exon sequences. The size of thenucleotide sequences (a) and (c) ranges from 1 to 50 kb, preferably from1 to 10 kb, more preferably from 2 to 6 kb, and most preferably from 2to 4 kb.

Methods of making a transgenic mouse comprising disruption in a selectedgene are well known to those of ordinary skill in the art and have beenused to successfully inactivate a wide range of genes.

Rat

Analogous or alternative [see, e.g., Zan et al, Nature Biotechnology(2003) 21:645-51; the disclosure of which is hereby incorporated byreference in its entirety] methods may be used to make a transgenic ratcomprising a disruption in a GPR50 gene.

Pig

Analogous or alternative methods may be used to make a transgenic pigcomprising a disruption in a GPR50 gene [see, e.g., Lai et al., Science(2002) 295:1089-1092; the disclosure of which is hereby incorporated byreference in its entirety].

Example 11 Endogenous GPR50 Exhibits Constitutive Activity forDecreasing a Level of Intracellular cAMP

Thyroid-stimulating hormone (TSH, or thyrotropin) receptor (TSHR) causesthe accumulation of intracellular cAMP on activation by its ligand TSH.An effective technique for measuring the decrease in production of cAMPcorresponding to activation of a receptor such as GPR50 is toco-transfect TSHR with GPR50 and to carry out the assay in the presenceof TSH to raise the level of basal cAMP, whereby TSHR acts as a “signalwindow enhancer.” Such an approach was used here.

Human HEK293 cells were co-transfected with thyroid-stimulating hormone(TSH, or thyrotropin) receptor (TSHR) and either pCMV vector or a cDNAplasmid encoding endogenous GPR50. Transfection was carried out usingLipofectamine (lnvitrogen). Forty-eight hours after transfection, thecells were stimulated with various concentrations of niacin and 100 nMTSH (Sigma) for 1 h before whole cell cAMP was determined using theAdenylyl Cyclase Flashplate Assay kit from Perkin Elmer catalog#:SMP004B], as described below.

The transfected cells were placed into anti-cAMP antibody-coated wellsthat contained 100 nM TSH and either niacin at various concentrations orvehicle. All conditions were tested in triplicate. After a 1 hincubation at room temperature to allow for stimulation of cAMP, aDetection Mix (provided in the Perkin Elmer kit) containing ¹²⁵I-cAMPwas added to each well and the plate was allowed to incubate for anotherhour at room temperature. The wells were then aspirated to removeunbound ¹²⁵I-cAMP. Bound ¹²⁵I-cAMP was detected using a Wallac MicrobetaCounter. The amount of cAMP in each sample was determined by comparisonto a standard curve, obtained by placing known concentrations of cAMP insome wells on the plate. Results are presented in FIG. 2.

As shown in FIG. 2, endogenous GPR50 is detectably constitutivelyactive, exhibiting constitutive activity for decreasing a level ofintracellular cAMP.

Example 12 Establishment of GPR50-Knockout (“Deficient”) Mice

GPR50-knockout (“deficient”) mice were established as described here.

A targeting vector was generated by inactivating the entire exon 2 andflanking intronic sequences and replacing it by a neomycin resistantcassette (see, FIG. 3A). In FIG. 3A, nucleotide numbering shown in smallitalics represents chromosome location according to Mouse Geneview:world wide web at ensembl.org/Mus_musculus/index.html.

The targeting vector was inserted into C57B1/6J embryonic stem (ES)cells by electroporation. Positive clones were isolated and exon 2deletion was verified by Southern blot. To check targeting at the 3′end, Southern blot analysis was performed on EcoRV-digested genomic DNAfrom ES cells with a 854-bp DNA fragment (“3′ external probe” as shownin FIG. 3A). Correctly targeted cells demonstrated a ˜7.8 kb modifiedband (due to the introduction of an EcoRV site at the 5′ end of the 3′arm). The 15.9 kb wild-type band was absent from targeted clones sinceGPR50 is X-linked.

To further verify that the exon 2 sequence had been deleted, Southernblot analysis was performed on EcoRI-digested genomic ES cells DNA witha 1031 bp DNA fragment (“internal probe” as shown in FIG. 3A) thatcontained most of the exon 2 sequence. As GPR50 is X-linked, no band wasdetected in correctly targeted clones, whereas a 7.1 kb band wasdetected in wild-type cells. All Southern blots were performed followingpreviously published methods [see, e.g., Sambrook et al, MolecularCloning: a Laboratory Manual (1989) Second Edition, Cold Spring Harbor,NY: CSH Laboratory Press; the disclosure of which is herein incorporatedby reference in its entirety].

Targeted and karyotypically normal ES cells were then microinjected intoBalb/c I/B and the injected blastocysts were transferred into the uteriof surrogate C57B1/6 mother mice. Male chimeras were bred to femaleC57B1/6J to generate F1 mice and germline transmission of GPR50 exon 2deletion was finer verified by PCR on mouse tail genomic DNA.

Five specific primer sets from different areas of GPR50 genomic sequencewere generated (“#1” to “#5” as shown in FIG. 3A). Primer sequences areshown in Table D below. Only primers #3-#5 should be detected inknockout mice, whereas all primers except #5 should be detected inwild-type mice. Tail were cut from mice and genomic DNA isolated usingphenol/chloroform extraction according to Sambrook et al 1989 (supra).PCR reaction was performed using the PCR Supermix from Invitrogen(Carlsbad, Calif.), 200 ng/μl of each of the forward and reverse primersand 100-500 ng genomic DNA in a reaction volume of 50 μl. The PCRproducts were run on 1.8% agarose gel to check for the presence orabsence of the amplified product for each primer pair and and to verifythe expected size. Results are shown in FIG. 3B.

Results shown in FIG. 3B confirm that GPR50-knockout (“deficient”) micewere established.

That GPR50-knockout (“deficient”) mice were established was alsoconfirmed by in situ hybridization analysis of GPR50 expression inhypothalamus.

TABLE D Primer Set Primer Primer Sequence #1 Forward PrimerTGCCATCAACCGTTACTGCTAC Exon 2 (SEQ ID NO: 18) (451 bp) Reverse PrimerGGGGATCTTGCCTGCCATTT (SEQ ID NO: 19) #2 Forward PrimerGCTCGTGCCTGTGTCGCTGTG Exon 2 (SEQ ID NO: 20) (511 bp) Reverse PrimerCAAGGCAATGGGAGGCTGAGA (SEQ ID NO: 21) #3 Forward PrimerCATTCGGCTGCATTGGCTGTAA Exon 1 (SEQ ID NO: 22) (418 bp) Reverse PrimerACTCCGTTCCTGTGGCGACTTC (SEQ ID NO: 23) #4 Forward PrimerGCAGGGTGGGCTCATCTTAGGTAT 3′ Intron (SEQ ID NO: 24) (518 bp) ReversePrimer TCTGGGATTTTGGGCTTGATGTGT (SEQ ID NO: 25) #5 Forward PrimerGGGCGCCCGGTTCTTTTTG Neo (SEQ ID NO: 26) cassette Reverse PrimerACACCCAGCCGGCCACAGTCG (515 bp) (SEQ ID NO: 27)

Example 13 Body Weight of GPR50-Knockout Mice on High Fat Diet or ChowCompared to Wild-Type Mice on High Fat Diet

An effect of GPR50 activity on weight gain induced by a high fat diet isshown in FIG. 4. Three groups of 30 week old male mice were housedindividually and allowed free access to water and food. The mice weremaintained on a 12 hour artificial light/12 h dark cycle and kept underconstant humidity (70%) and temperature (22° C.) conditions. A firstgroup of wild-type C57B1/6J mice (n=6) (“WT” in FIG. 4) and a secondgroup of GPR50-knockout mice (n=2) (“KO” in FIG. 4) were allowed freeaccess to high fat diet (“HFD” in FIG. 4) (D12266B, Research Diet, 31.8%fat/Kcal), while a third group of GPR50-knockout mice (n=3) were allowedaccess to chow pellets (“Chow” in FIG. 4) (Teklab 8604, 4.4% fat) for 15weeks.

Body weight is shown in FIG. 4A as mean±SEM. Body weight gain as apercentage of initial body weight is shown in FIG. 4B as mean±SEM. It isapparent from inspection of FIG. 4 (for example, comparison of theGPR50-knockout mice on the high fat diet with the wild-type C57B1/6Jmice on the high fat diet) that loss of GPR50 activity in theGPR50-knockout mice conferred protection from (that is, decreased)weight gain induced by a high fat diet.

An analogous experiment carried out in 70 wk old male GPR50-knockout(“KO”) (n=6) and wild-type littermate (“WT”) (n=6) mice on the high fatdiet (D12266B, Research Diet, 31.8% fat/Kcal) for three weeks is shownin FIG. 5.

Example 14 In Vivo Effects of a GPR50 Inverse Agonist or Antagonist onIncreased Adiposity Induced by a High-Fat Diet in Mice

An inverse agonist or antagonist of GPR50 can be shown to conferprotection from increased adiposity induced by a high fat diet. Twogroups of age- and sex-matched 5-30 week old wild-type C57B1/6J mice arehoused individually and allowed free access to water and food. The miceare maintained on a 12 hour artificial light/12 h dark cycle and keptunder constant humidity (70%) and temperature (22° C.) conditions. Miceare allowed free access to high fat diet (D12266B, Research Diet, 31.8%fat/Kcal), for aperiod of4-15 weeks. Over the course of the 4-15 weekperiod, an inverse agonist or antagonist of GPR50 having inverse agonistor antagonist activity at mouse GPR50 or vehicle alone is injected dailyinto the tail vein. A preferred dose of the GPR50 inverse agonist orantagonist is 0.1-100 mg/kg. Other preferred dose is selected from thegroup consisting of 0.1 mg/kg, 0.3 mg/kg, 1.0 mg/kg, 3.0 mg/kg, 10mg/kg, 30 mg/kg and 100 mg/kg.

At the conclusion of the 4-15 week period, the mice are euthanized byCO₂ inhalation, and the epididymal and inguinal footpads are harvestedand weighed as a measure of adiposity. The results can demonstrate thatthe GPR50 inverse agonist or antagonist confers protection from (thatis, decreases) the increased adiposity (increased weight of theepididymal and inguinal footpads) induced by a high fat diet.

It is expressly contemplated that the GPR50 inverse agonist orantagonist can be a selective GPR50 inverse agonist or antagonist. It isexpressly contemplated that a high fat diet having less or more than31.8% fat/Kcal can be used. It is expressly contemplated thatadministration of the inverse agonist or antagonist can be other thanintravenous, for example that administration of the inverse agonist orantagonist can be intraperitoneal or oral. It is expressly contemplatedthat mice younger than 5 weeks or older than 30 weeks can be used. It isexpressly contemplated that the period of injection can be less than 4weeks or longer than 15 weeks. It is expressly contemplated that anon-human mammal other than mouse can be used, for example rat.

Example 15 In Vivo Effects of a GPR50 Inverse Agonist or Antagonist onIncreased Percentage Body Fat Induced by a High-Fat Diet in Mice

An inverse agonist or antagonist of GPR50 can be shown to conferprotection from increased percentage body fat induced by a high fatdiet. Two groups of age- and sex-matched 5-30 week old wild-typeC57B1/6J mice are housed individually and allowed free access to waterand food. The mice are maintained on a 12 hour artificial light/12 hdark cycle and kept under constant humidity (70%) and temperature (22°C.) conditions. Mice are allowed free access to high fat diet (D12266B,Research Diet, 31.8% fat/Kcal), for a period of 4-15 weeks. Over thecourse of the 4-15 week period, an inverse agonist or antagonist ofGPR50 having inverse agonist or antagonist activity at mouse GPR50 orvehicle alone is injected daily into the tail vein. A preferred dose ofthe GPR50 inverse agonist or antagonist is 0.1-100 mg/kg. Otherpreferred dose is selected from the group consisting of 0.1 mg/kg, 0.3mg/kg, 1.0 mg/kg, 3.0 mg/kg, 10 mg/kg, 30 mg/kg and 100 mg/kg.

At the conclusion of the 4-15 week period, the mice are euthanized byCO₂ inhalation and percentage body fat is evaluated by determining bodycomposition by densitometry using Dual energy X-ray absorptiometry(DEXA) (Lunar PIXImus, Lunar PIXImus Corp., Madison, Wis.). The data areanalyzed using Lunar PIXImus 2.2.0 software according to themanufacturer's instructions. The results can demonstrate that the GPR50inverse agonist or antagonist confers protection from (that is,decreases) the increased percentage body fat induced by a high fat diet.

It is expressly contemplated that the GPR50 inverse agonist orantagonist can be a selective GPR50 inverse agonist or antagonist. It isexpressly contemplated that a high fat diet having less or more than31.8% fat/Kcal can be used. It is expressly contemplated thatadministration of the inverse agonist or antagonist can be other thanintravenous, for example that administration of the inverse agonist orantagonist can be intraperitoneal or oral. It is expressly contemplatedthat mice younger than 5 weeks or older than 30 weeks can be used. It isexpressly contemplated that the period of injection can be less than 4weeks or longer than 15 weeks. It is expressly contemplated that anon-human mammal other than mouse can be used, for example rat.

Example 16 In Vivo Effects of a GPR50 Inverse Agonist or Antagonist onWeight Gain Induced by a High-Fat Diet in Mice

An inverse agonist or antagonist of GPR50 can be shown to conferprotection from weight gain induced by a high fat diet. Two groups ofage- and sex-matched 5-30 week old wild-type C57B1/6J mice are housedindividually and allowed free access to water and food. The mice aremaintained on a 12 hour artificial light/12 h dark cycle and kept underconstant humidity (70%) and temperature (22° C.) conditions. Mice areallowed free access to high fat diet (D12266B, Research Diet, 31.8%fat/Kcal), for a period of 4-15 weeks. Over the course of the 4-15 weekperiod, an inverse agonist or antagonist of GPR50 having inverse agonistor antagonist activity at mouse GPR50 or vehicle alone is injected dailyinto the tail vein. A preferred dose of the GPR50 inverse agonist orantagonist is 0.1-100 mg/kg. Other preferred dose is selected from thegroup consisting of 0.1 mg/kg, 0.3 mg/kg, 1.0 mg/kg, 3.0 mg/kg, 10mg/kg, 30 mg/kg and 100 mg/kg.

At weekly intervals over the course of the 4-15 week period, the miceare weighed. The results can demonstrate that the GPR50 inverse agonistor antagonist confers protection from (that is, decreases) weight gaininduced by a high fat diet.

It is expressly contemplated that the GPR50 inverse agonist orantagonist can be a selective GPR50 inverse agonist or antagonist. It isexpressly contemplated that a high fat diet having less or more than31.8% fat/Kcal can be used. It is expressly contemplated thatadministration of the inverse agonist or antagonist can be other thanintravenous, for example that administration of the inverse agonist orantagonist can be intraperitoneal or oral. It is expressly contemplatedthat mice younger than 5 weeks or older than 30 weeks can be used. It isexpressly contemplated that the period of injection can be less than 4weeks or longer than 15 weeks. It is expressly contemplated that anon-human mammal other than mouse can be used, for example rat.

Example 17 Analysis of Co-Expression of GPR50 by NPY Neurons in theCentral Part of the Dorsomedial Nucleus of the Hypothalamus (DMHc) inRat

Co-expression of GPR50 by neuropeptide-Y (NPY) neurons in the centralpart of the dorsomedial nucleus of the hypothalamus (DMHc) in rat wasinvestigated by in situ hybridization, using radiolabeled antisenseprobe for GPR50 in combination with a digoxigenin (Dig)-labeledantisense probe for NPY. Rat GPR50 probe corresponding to nucleotides397 to 918 of SEQ ID NO: 7 was inserted into pBS vector (Stratagene, LaJolla, Calif.). Rat NPY probe corresponding to rat NPY cDNA sequence(511 nucleotides) spanning the coding sequence (see, e.g., GenBank®Accession No. NM_(—)012614) was inserted into pBS vector (Stratagene).In situ hybridization was carried out essentially as described below.

Rats were killed by rapid decapitation 1-2 h after initiation of thelight cycle. Brains were removed, frozen in isopentane (−40° C.), andstored at −80° C. Serial 12-μm sections from the central part of thedorsomedial nucleus of the hypothalamus (DMHc) were prepared on acryostat, thaw-mounted onto polylysine-subbed slides, and stored at −80°C. until processing.

Sense and antisense ³³P radiolabeled probes were generated by in vitrotranscription by incubating linearized plasmids in transcription buffercontaining RNasin (40 units), DYT (2 mM), ATP, CTP and GTP (0.33 mM),[α-³³P]-UTP (Perkin Elmer, 50 μCi, NEG307 H001MC) and the appropriatepolymerase (T7 50 units or T3 20 units). Probes were DNase treated,purified by ethanol precipitation and resuspend in 2× hybridizationbuffer (8×SET, 2×Denhardt's, 0.4% SDS, 200 mM dithiothreitol (DTT), 500ug/ml tRNA, 50 ug/ml polyA, 50 ug/ml polyC).

Antisense digoxigenin labeled probes were generated by in vitrotranscription by incubating linearized plasmids in transcription buffercontaining RNasin (40 units), DTT (2 mM), nucleotide mix containingdigoxigenin labeled UTP (rNTP digoxigenin RNA labeling mix, Roche#1277073) and the appropriate polymerase (T7 50 units or T3 20 units).Probes were DNase treated and cleaned up through a centrisep column(Princeton Separations, #CS-901).

Tissue sections were removed from the freezer and allow to air dry for15 min. Sections were subsequently fixed in 4% paraformaldehyde inphosphate buffer (0.1 M, pH 7.4) for 30 min at room temperature, rinsed3 times in 1×PBS, and acetylated in 0.1M triethanolamine (TEA), pH 8.0for 2 min then briefly in the same buffer containing 0.25% aceticanhydride. Slides were then rinsed for 5 minutes in 1×PBS and thendehydrated through graded alcohol concentrations and air dried.Radiolabeled probes were diluted in 2× hybridization buffer to yield anapproximate concentration of 16×10⁶ cpm per slide. Salmon sperm wasadded at a final concentration of 20 ug/slide and digoxigenin labeledprobe was added to a final concentration of 500 ng/slide. Dextransulfate/Formamide (20%) was added to give a 1:1 ratio with 2×hybridization buffer. Diluted probe was placed on slides, coverslippedand were incubated at 55° C. for 16-18 hours in plastic trays humidifiedwith 1×PBS. Coverslips were floated off with 1 mM DTT/4×SSC (600 nMsodium chloride and 60 mM sodium citrate, pH 7.2) and sections weresubsequently washed once in 4×SSC for 10 min, incubated in ribonucleaseA (200 ug/ml) for 60 min in a 37° C. water bath, then rinsed in 2×, 1×,and 0.5×SSC for 5 minutes each. Sections were washed to a finalstringency of 0.1×SSC at 65° C. for 1 hour, then washed twice in 0.1×SSCthen washed in TN (100 mM Tris, pH 7.5, 150 mM NaCl) for 5 minutes.Sections were then placed in 0.5% Casein/TN blocking solution for 30minutes then incubated for 2 hours with anti-digoxigenin-AP antibody(Roche, #1093274) diluted 1:300 in 0.5% Casein/TN solution. Sectionswere then washed 3 times, 2 minutes each in TN and then 3 times 5minutes each in TNM (100 mM Tris, pH 9.5, 100 mM NaCl, 50 mM MgCl₂).After the last wash, sections were incubated in color reaction (0.2mg/ml levamisole, 3.4 ul/ml NBT (Roche #1383213), 3.5 ul/ml BCIP (Roche#1383221) in TNM and 0.22 u sterile filtered) for 20-30 minutes andreaction stopped in TE for 30 minutes. Antibody was striped off byincubating sections in 0.1M glycine and 0.5% triton-X 100 for 10 minutesand washed in water. Sections were fixed in 2.5% glutaraldehyde for 1-2hours and washed with water then air dried. Once section dried, theywere exposed to x-ray sensitive film (Bio-Max, Kodak, Eastman Kodak Co.,Rochester, N.Y.) for 2-7 days and dipped in photographic emulsion(Ilford Scientific K.5D Emulsion in gel form from Polysciences, #17537)dried and stored in slides box with desiccant at 4° C. for 4-8 weeksdepending on the level of expression. After development of dipped slidesfollowing manufacturer recommendations (Kodak D19), sections were washedextensively in water and air dried then mounted with coverslips formicroscopic examination.

Images of the distribution of GPR50 and NPY mRNA-containing cells wereobtained using an Olympus BX51 microscope connected to a videocamera(NTSC 750CE) using Stereoinvestigator® v6.55.2 software(Microbrightfield, Vt.). Nonradioactive riboprobes were visualized underbrightfield as a purple precipitate, and radioactive probes werevisualized under darkfield by silver grain distribution.

A representative photomicrographic image illustrating the expression ofGPR50 and NPY in the central part of the dorsomedial nucleus of thehypothalamus (DMHc) in rat is presented in FIG. 6A. Note the presence ofneurons expressing only GPR50 (open arrowhead), neurons expressing onlyNPY (solid arrowhead), and neurons co-expressing GPR50 and NPY (arrow).

The percentage of NPY neurons in rat DMHc co-expressing GPR50 (“doubleGPR50/NPY”) was estimated by analysis of tissue sections from each oftwo rats, as shown in FIG. 6B. For each rat, analysis was carried out ontwo consecutive sections a and b at position 1 and on two consecutivesections a and b at position 2, with position 1 and position 2 beingseparated by 120 microns. The percentage of NPY neurons co-expressingGPR50 was taken as the average±SEM of the percentages for the individualslides and was determined to be 54.3±2.8, as set forth in FIG. 6B.

Example 18 Yeast Reporter Assay for GPR50 Modulator (e.g., InverseAgonist or Antagonist) Activity

The yeast cell-based reporter assays have previously been described inthe literature (e.g., see Miret et al, J Biol Chem (2002) 277:6881-6887;Campbell et al, Bioorg Med Chem Lett (1999) 9:2413-2418; King et al,Science (1990) 250:121-123; WO 99/14344; WO 00/12704; and U.S. Pat. No.6,100,042). Briefly, yeast cells have been engineered such that theendogenous yeast G-alpha (GPA1) has been deleted and replaced withG-protein chimeras constructed using multiple techniques. Additionally,the endogenous yeast alpha-cell GPCR, Ste3 has been deleted to allow fora homologous expression of a mammalian GPCR of choice. In the yeast,elements of the pheromone signaling transduction pathway, which areconserved in eukaryotic cells (for example, the mitogen-activatedprotein kinase pathway), drive the expression of Fus1. By placingβ-galactosidase (LacZ) under the control of the Fus1 promoter (Fus1p), asystem has been developed whereby receptor activation leads to anenzymatic readout.

Yeast cells are transformed by an adaptation of the lithium acetatemethod described by Agatep et al (Agatep et al, 1998, Transformation ofSaccharomyces cerevisiae by the lithium acetate/single-stranded carrierDNA/polyethylene glycol (LiAc/ss-DNA/PEG) protocol. Technical TipsOnline, Trends Journals, Elsevier). Briefly, yeast cells are grownovernight on yeast tryptone plates (YT). Carrier single-stranded DNA (10μg), 2 μg of each of two Fus1p-LacZ reporter plasmids (one with URAselection marker and one with TRP), 2 μg of GPR50 (e.g., human receptor)in yeast expression vector (2 μg origin of replication) and a lithiumacetate/polyethylene glycol/TE buffer is pipetted into an Eppendorftube. The yeast expression plasmid containing the receptor/no receptorcontrol has a LEU marker. Yeast cells are inoculated into this mixtureand the reaction proceeds at 30° C. for 60 min. The yeast cells are thenheat-shocked at 42° C. for 15 min. The cells are then washed and spreadon selection plates. The selection plates are synthetic defined yeastmedia minus LEU, URA and TRP (SD-LUT). After incubating at 30° C. for2-3 days, colonies that grow on the selection plates are then tested inthe LacZ assay.

In order to perform fluorimetric enzyme assays for β-galactosidase,yeast cells carrying the subject GPR50 receptor are grown overnight inliquid SD-LUT medium to an unsaturated concentration (i.e. the cells arestill dividing and have not yet reached stationary phase). They arediluted in fresh medium to an optimal assay concentration and 90 μl ofyeast cells are added to 96-well black polystyrene plates (Costar). Testcompounds, dissolved in DMSO and diluted in a 10% DMSO solution to 10×concentration, are added to the plates and the plates placed at 30° C.for 4 h. After 4 h, the substrate for the β-galactosidase is added toeach well. In these experiments, Fluorescein di(β-D-galactopyranoside)is used (FDG), a substrate for the enzyme that releases fluorescein,allowing a fluorimetric read-out. 20 μl per well of 500 μM FDG/2.5%Triton X100 is added (the detergent is necessary to render the cellspermeable). After incubation of the cells with the substrate for 60 min,20 μl per well of 1M sodium carbonate is added to terminate the reactionand enhance the fluorescent signal. The plates are then read in afluorimeter at 485/535 nm.

A decrease in fluorescent signal in GPR50-transformed yeast cells overthat in yeast cells transformed with empty vector is indicative of atest compound being a compound that inhibits GPR50 receptorfunctionality (e.g., a compound that is an inverse agonist or antagonistof GPR50). In certain embodiments, compounds of the invention give adecrease in fluorescent signal below that of the background signal (thesignal obtained in the presence of vehicle alone).

An increase in fluorescent signal in GPR50-transformed yeast cells overthat in yeast cells transformed with empty vector is indicative of atest compound being a compound that stimulates GPR50 receptorfunctionality (e.g., a compound that is an agonist or partial agonist ofGPR50). In certain embodiments, compounds of the invention give anincrease in fluorescent signal above that of the background signal (thesignal obtained in the presence of vehicle alone).

Example 19 Receptor Binding Assay

A test compound can be evaluated for its ability to reduce formation ofthe complex 35 between a compound known to be a ligand of a Gprotein-coupled receptor of the invention and the receptor. In certainembodiments, the known ligand is radiolabeled. The radiolabeled knownligand can be used in a screening assay to identify/evaluate compounds.In general terms, a newly synthesized or identified compound (i.e., testcompound) can be evaluated for its ability to reduce binding of theradiolabeled known ligand to the receptor, by its ability to reduceformation of the complex between the radiolabeled known ligand and thereceptor.

In other aspect, a test compound can be radiolabeled and shown to be aligand of a subject GPCR of the invention by evaluating its ability tobind to a cell comprising the subject GPCR or to membrane comprising thesubject GPCR.

A level of specific binding of the radiolabled known ligand in thepresence of the test compound less than a level of specific binding ofthe radiolabeled known ligand in the absence of the test compound isindicative of less of the complex between said radiolabeled known ligandand said receptor being formed in the presence of the test compound thanin the absence of the test compound.

Assay Protocol for Detecting the Complex Between a Compound Known to bea Ligand of a G Protein-Coupled Receptor of the Invention and theReceptor

A. Preparation of the Receptor

293 cells are transiently transfected with 10 ug expression vectorcomprising a polynucleotide encoding a G protein-coupled receptor of theinvention using 60 ul Lipofectamine (per 15-cm dish). The transientlytransfected cells are grown in the dish for 24 hours (75% confluency)with a media change and removed with 10 ml/dish of Hepes-EDTA buffer (20mM Hepes+10 nM EDTA, pH 7.4). The cells are then centrifuged in aBeckman Coulter centrifuge for 20 minutes, 17,000 rpm (JA-25.50 rotor).Subsequently, the pellet is resuspended in 20 mM Hepes+1 mM EDTA, pH 7.4and homogenized with a 50-ml Dounce homogenizer and again centrifuged.After removing the supernatant, the pellets are stored at −80° C., untilused in binding assay. When used in the assay, membranes are thawed onice for 20 minutes and then 10 mL of incubation buffer (20 mM Hepes, 1mM MgCl₂, 100 mM NaCl, pH 7.4) added. The membranes are then vortexed toresuspend the crude membrane pellet and homogenized with a BrinkmannPT-3100 Polytron homogenizer for 15 seconds at setting 6. Theconcentration of membrane protein is determined using the BRL Bradfordprotein assay.

B. Binding Assay

For total binding, a total volume of 50 ul of appropriately dilutedmembranes (diluted in assay buffer containing 50 mM Tris HCl (pH 7.4),10 mM MgCl₂, and 1 mM EDTA; 5-50 ug protein) is added to 96-wellpolypropylene microtiter plates followed by addition of 100 ul of assaybuffer and 50 ul of a radiolabeled known ligand. For nonspecificbinding, 50 ul of assay buffer is added instead of 100 ul and anadditional 50 ul of 10 uM said known ligand which is not radiolabeled isadded before 50 ul of said radiolabeled known ligand is added. Platesare then incubated at room temperature for 60-120 minutes. The bindingreaction is terminated by filtering assay plates through a MicroplateDevices GF/C Unifilter filtration plate with a Brandell 96-well plateharvestor followed by washing with cold 50 mM Tris HCl, pH 7.4containing 0.9% NaCl. Then, the bottom of the filtration plate aresealed, 50 ul of Optiphase Supermix is added to each well, the top ofthe plates are sealed, and plates are counted in a Trilux MicroBetascintillation counter. For determining whether less of the complexbetween said radiolabeled known ligand and said receptor is formed inthe presence of a test compound, instead of adding 100 ul of assaybuffer, 100 ul of appropriately diluted said test compound is added toappropriate wells followed by addition of 50 ul of said radiolabledknown ligand.

Example 20 Analysis of the Effect of Food Restriction on the Expressionof GPR50 in the Central Part of the Dorsomedial Nucleus of theHypothalamus (DMHc) in Rat

The effect of food restriction on the expression of GPR50 in the centralpart of the dorsomedial nucleus of the hypothalamus (DMHc) in rat wasdetermined by in situ hybridization. The effect of food restriction onthe expression of neuropeptide-Y (NPY), an orexigenic molecule, was alsodetermined. Tissue sections of the DMHc were prepared essentially asdescribed supra in Example 17, as was in situ hybridization carried outusing ³³P radiolabeled antisense probe.

Twenty week old male Sprague Dawley rats were divided into two groups ofseven rats each, housed in cages singly. One group, the ad libitum fedgroup, was provided an unrestricted amount of food (Teklab 8604, 4.4%fat) each day over a period of twelve days. A second group, thefood-restricted group, was provided 16 g of the same food each day (70%of the average daily food intake of the rats under ad libitumconditions) over the same twelve day period. Food was provided in themorning. The amount of food intake in the rats during the previous 24hour period also was determined in the morning. The weight of the ratsalso was determined in the morning. The food intake in the ad libitumfed and in the food-restricted rats is shown in the upper panel of FIG.7A. The percentage of original body weight in the rats (that is, thebody weight of the rat expressed as the percentage of its body weight atthe initiation of the experiment, Day 0) is shown in the lower panel ofFIG. 7A. From inspection of FIG. 7A, it is apparent that foodrestriction evidenced as declining body weight in the food-restrictedrats.

At the end of the twelve day period, the rats were sacrificed. Tissuesections of the DMHc were prepared and subjected to in situhybridization with ³³P radiolabeled GPR50 antisense probe or with ³³Pradiolabeled NPY antisense probe. Sense probe was used as a specificitycontrol. Bound radioactive probe was visualized under darkfield bysilver grain distribution. Representative photomicrographic images fromtwo different rats in the ad libitum fed group and from two differentrats in the food-restricted group are shown in FIG. 7B for GPR50. Frominspection of FIG. 7B, it is apparent that food restriction led tomarked up-regulation of GPR50 expression in the DMHc.

The relative levels of GPR50 mRNA expression in the DMHc for the adlibitum fed and food-restricted groups as evidenced by the in situhybridization were determined using Scion Image Version 1.63 analysis ofautoradiogram optical density, and the results are presented in thelower panel of FIG. 7C. The relative levels of NPY mRNA expression inthe DMHc were likewise determined for the ad libitum fed andfood-restricted groups, and the results are presented in the upper panelof FIG. 7C. From inspection of FIG. 7C, it is apparent that foodrestriction led to marked up-regulation of GPR50 expression in the DMHcand that this up-regulated GPR50 expression was associated in the DMHcwith up-regulated expression of NPY.

1. A method of identifying a candidate compound as a modulator of bodymass or adiposity in a mammal, comprising the steps of: (a) contactingthe candidate compound with a G protein-coupled receptor (GPCR)comprising an amino acid sequence having at least 80% identity to SEQ IDNO: 2; wherein the receptor couples to a G protein; (b) determining theability of the compound to inhibit or stimulate functionality of theGPCR; (c) administering a compound which inhibits or stimulatesfunctionality of the GPCR in step (b) to a mammal; and (d) determiningwhether the compound modulates body mass or adiposity in said mammal. 2.The method of claim 1, wherein step b) of the method comprises detectinga second messenger.
 3. The method of claim 1, wherein said determiningstep b) is by a process comprising the use of a melanophore assay or bya process comprising the measurement of GTPyS binding to a membranecomprising the GPCR.
 4. The method of claim 1, wherein said contactingof the candidate compound with a GPCR comprises contacting the candidatecompound with a eukaryotic host cell comprising the GPCR or withmembrane thereof that comprises the GPCR.
 5. The method of claim 1,wherein the method further comprises formulating said compound as apharmaceutical composition.
 6. The method of claim 1, wherein said GPCRcomprises an amino acid sequence having at least 90% identity to SEQ IDNO:
 2. 7. The method of claim 1, wherein said GPCR comprises an aminoacid sequence having at least 95% identity to SEQ ID NO:
 2. 8. Themethod of claim 1, wherein said determining step d) comprises measuringthe weight of said mammal.
 9. The method of claim 1, wherein saiddetermining step d) comprises measuring weight gain induced by a highfat diet.
 10. The method of claim 1, wherein the compound inhibitsfunctionality of the GPCR.
 11. The method of claim 10, wherein thecompound is an antagonist of the GPCR.
 12. The method of claim 10,wherein the compound is an inverse agonist of the GPCR.
 13. The methodof claim 1, wherein the compound stimulates functionality of the GPCR.14. The method of claim 13, wherein the compound is an agonist of saidGPCR.
 15. The method of claim 13, wherein the compound is a partialagonist of said GPCR.